Chromatographic materials

ABSTRACT

The present invention provides a chromatographic stationary phase material various different types of chromatography. One example chromatographic stationary phase is represented by Formula 1 [X](W) a (Q) b (T) c  (Formula 1). X can be a high purity chromatographic core composition. W can be absent and/or can include hydrogen and/or can include hydroxyl on the surface of X. Q can be bound directly to X and can include a first hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte. T can be bound directly to X and can include a second hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte. Additionally, Q and T can essentially eliminate chromatographic interaction between the analyte, and X and W, thereby minimizing retention variation over time (drift or change) under chromatographic conditions utilizing low water concentrations.

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of priority to U.S. Provisional Application No. 61/647,303 filed May 15, 2012, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to chromatographic materials. The invention relates more particularly, in various embodiments, to chromatographic materials for normal phase chromatography, supercritical fluid chromatography, carbon dioxide based chromatography, hydrophobic interaction liquid chromatography, hydrophilic interaction liquid chromatography, subcritical fluid chromatography, high pressure liquid chromatography, and solvated gas chromatography that mitigate or avoid retention drift or change while exhibiting useful overall retention, as well as corresponding apparatuses, kits, methods of manufacture, and methods of use.

BACKGROUND OF THE INVENTION

Packing materials for fluid or liquid chromatography can be generally classified into two types: organic materials (e.g., polydivinylbenzene) and inorganic materials (e.g., silica). Many organic materials are chemically stable against strongly alkaline and strongly acidic mobile phases, allowing flexibility in the choice of mobile phase composition and pH. However, organic chromatographic materials can result in columns with low efficiency, particularly with low molecular-weight analytes. Many organic chromatographic materials not only lack the mechanical strength of typical chromatographic silica and also shrink and swell when the composition of the mobile phase is changed.

Silica is widely used in High Performance Liquid Chromatography (HPLC), Ultra High Performance Liquid Chromatography (UHPLC), and Supercritical Fluid Chromatography (SFC). Some applications employ silica that has been surface-derivatized with an organic functional group such as octadecyl (C18), octyl (C8), phenyl, amino, cyano, and the like. As stationary phases for HPLC, these packing materials can result in columns that have high efficiency and do not show evidence of shrinking or swelling.

Hybrid materials can provide solutions to certain chromatographic problems experienced with silica based packing materials. Hybrid materials can provide improvements including improved high and low pH stability, mechanical stability, peak shape when used at pH 7, efficiency, retentivity, and desirable chromatographic selectivity.

However, potential problems can exist for conventional hybrid materials and silica materials in other applications. One problem is poor peak shape for bases when used at low pH, which can negatively impact loadability and peak capacity when used at low pH. Another problem is a change in acidic and basic analyte retention times (denoted ‘drift’) after a column is exposed to repeated changes in mobile phase pH (e.g., switching repeatedly from pH 10 to 3).

Another problem is retention drift or change, for example in chromatography modes with little water (e.g., less than 5%, less than 1%). For example, retention drift or change is observed under standard SFC conditions for both silica and organic-inorganic hybrid (e.g., BEH Technology™ materials available from Waters Technologies Corporation, Milford Mass.) based chromatographic phases, bonded and unbonded. Other SFC stationary phases can also exhibit similar retention drift or change.

SUMMARY OF THE INVENTION

In various aspects and embodiments, the invention provides chromatographic materials for normal phase chromatography, supercritical fluid chromatography, carbon dioxide based chromatography, hydrophobic interaction liquid chromatography, hydrophilic interaction liquid chromatography, subcritical fluid chromatography, high pressure liquid chromatography, and solvated gas chromatography that mitigate or avoid retention drift or change while exhibiting useful overall retention, as well as corresponding apparatuses, kits, methods of manufacture, and methods of use.

The invention advantageously mitigates or avoids retention drift or change while exhibiting useful overall retention. For example, in SFC, retention drift or change can be (among various other theories) attributed to alkoxylation of solvent accessible silanols on the particle under the standard CO₂/MeOH mobile phase (and/or by other alcohol co-solvents) utilized for SFC. This is a problem, as users observe a change in the chromatography (e.g., retention time) obtained on their SFC system as the column ages, and again when a new, non-alkoxylated column is put on the system.

In various aspects and embodiments, the invention provides various solutions to such retention drift or change and related problems (e.g., retention, peak shape, and the like) through selection and/or modification of the chromatographic material.

For example, the invention includes specialized functionalization of a chromatographic core surface (e.g., with particular endcapping groups, and combinations thereof), which essentially prevent chromatographic interaction between and analyte and the chromatographic core surface, which maintaining desired interaction between the analyte and the chromatographic material. In one alternative example, the invention include pre-alkoxylating (or effectively pre-alkoxylating) a chromatographic core surface. In further alternative examples, the invention includes the use of different base material(s) that mitigate retention drift or change due to having less acidic silanols, a reduced number of silanols, a non-silica based material, or a combination thereof. The invention include various additional advantages, including but not limited to, the ability to selection/design selectivity through selection/design of the chemical modifications.

In one aspect, the present invention provides a chromatographic stationary phase material for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography. The chromatographic stationary phase is represented by Formula 1 [X](W)_(a)(Q)_(b)(T)_(c) (Formula 1). X can be a high purity chromatographic core composition having a surface including a silica core material, metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof. W can be absent and/or can include hydrogen and/or can include hydroxyl on the surface of X. Q can be bound directly to X and can include a first hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte. T can be bound directly to X and can include a second hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte. Additionally, Q and T can essentially eliminate chromatographic interaction between the analyte, and X and W, thereby minimizing retention variation over time (drift) under chromatographic conditions utilizing low water concentrations.

In another aspect, the present invention provides a chromatographic stationary phase represented by Formula 1: [X](W)_(a)(Q)_(b)(T)_(c) (Formula 1). X can be a chromatographic core material including a silica based, metal oxide based, or inorganic-organic hybrid based core surface. W can be absent and/or can include hydrogen and/or can include hydroxyl on the surface of X. Q can be bound directly to X and can include a first hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with an analyte. T can be bound directly to X and can include a second hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte. Additionally, Q and T can essentially eliminate chromatographic interactions between the analyte, and X and W.

In yet another aspect, the present invention provides a chromatographic stationary phase material represented by Formula 1:[X](W)_(a)(Q)_(b)(T)_(c) (Formula 1). X can be a chromatographic core material including a silica based, metal oxide based, or inorganic-organic hybrid based core surface. W can be absent and/or can include hydrogen and/or can include hydroxyl on the surface of X. Q and T can each be independently bound directly to X. Q can include a hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with an analyte. X can include a non-polar group, or X can include a hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with an analyte. Q can include a non-polar group. Additionally, Q and T can essentially eliminate chromatographic interaction between the analyte, and X and W.

In one or more embodiments of any of the above aspects, Q can be represented by

In some embodiments, n¹ is an integer from 0-30; n² is an integer from 0-30; and n³=0 or 1, provided that when n³=0, n¹ is not 0. Each occurrence of R¹, R², R³ and R⁴ can independently represent hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, a protected or deprotected alcohol, a zwitterion, or a group Z. In some embodiments, Z represents a surface attachment group having the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein X is an integer from 1-3, y is an integer from 0-2, Z is an integer from 0-2, and x+y+z=3. Each occurrence of R⁵ and R⁶ can independently represent methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group. B¹ can represent a siloxane bond. In some embodiments, Z represents an attachment to a surface organofunctional hybrid group through a direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane linkage. In some embodiments, Z represents an adsorbed, surface group that is not covalently attached to the surface of the material. In some embodiments, Y is an embedded polar functionality. In some embodiments, A represents a hydrophilic terminal group. In some embodiments, A represents hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, or group Z. In some embodiments, A represents a functionalizable group.

In one or more embodiments of any of the above aspects, T is represented by:

In some embodiments, n¹ is an integer from 0-5; n² is an integer from 0-5; and n³=0 or 1, provided that when n³=0, n¹ is not 0. Each occurrence of R¹, R², R³ and R⁴ can independently represent hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, a protected or deprotected alcohol, a zwitterion, or a group Z. In some embodiments, Z represents a surface attachment group having the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein X is an integer from 1-3, y is an integer from 0-2, Z is an integer from 0-2, and x+y+z=3. Each occurrence of R⁵ and R⁶ can independently represent methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group and B¹ can represent a siloxane bond. In some embodiments, Z represents an attachment to a surface organofunctional hybrid group through a direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane linkage. In some embodiments, Z represents an adsorbed, surface group that is not covalently attached to the surface of the material. In some embodiments, Z represents a silyl ether bond. In some embodiments, Y is an embedded polar functionality. Additionally, in some embodiments, A represents a hydrophilic or ionizable terminal group; or in other embodiments A represents hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, or group Z.

In one or more embodiments of any of the above aspects, the chromatographic stationary T is one of the following structures:

In one or more embodiments, R⁵ and R⁶ independently represent methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group. Additionally, in some embodiments, A, A₁, and A₂ are independently selected hydrophilic/ionizable groups—including cyano, hydroxyl, fluoro, trifluoro, substituted aryl, ester, ether, amide, carbamate, urea, sulfoxide, nitro, nitroso, boronic acid, boronic ester, urea, thioether, sulfonyl, thiourea, thiocarbonate, thiocarbarnate, ethylene glycol, heterocyclic, methyl, ethyl, n-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower a protected or deprotected alcohol, or a zwitterion group, or triazole functionalities. In other embodiments, A, A₁ and A₂ are independently selected from non-polar groups—including methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, or lower alkyl. In yet other embodiments, A is independently selected from a hydrophilic/ionizable group, and A₁ is independently selected from a non-polar group and is independently selected from either a hydrophilic/ionizable group or from a non-polar group.

In one or more embodiments, Q and T are different. In other embodiments, Q and T are the same. In some embodiments, the first functional group includes a diol, trimethoxysilyl ethyl pyridine, diethylaminotrimethoxysilane, a sulfur, nitrogen or oxygen based polar silanes carbonate, carbamate, amide, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocycle, or triazole. The functional group included by T can be an amine, trimethoxysilyl ethyl pyridine, diethylaminotrimethoxysilane, a sulfur, nitrogen or oxygen based polar silanes carbonate, carbamate, amide, urea, ether, thioether, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocycle, or triazole. Additionally, T, Q, or both Q and T can be a chiral functional group adapted for a chiral separation.

In some embodiments, Q is one of the following structures:

In some embodiments, Q comprises a borate or nitro functional group.

In some embodiments, Z is a surface attachment group having the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein x is an integer from 1-3, y is an integer from 0-2, z is an integer from 0-2, and x+y+z=3. Each occurrence of R⁵ and R⁶ can independently represents methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group B¹ can represent a siloxane bond. In some embodiments, Z is an attachment to a surface organofunctional hybrid group through a direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane linkage. Additionally, in some embodiments, Z is an adsorbed, surface group that is not covalently attached to the surface of the material.

In some embodiments, the ratio b/c is about 0.05-75, 0.05-50, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90. Additionally, in one or more embodiments b=c, or b=0 and c>0 or b>0 and c=0. In some embodiments, a is ≧0. In some embodiments, the combined surface coverage is greater than about 1, 2, 3, 3.1, 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, or 8 μmol/m². The combined surface coverage can be greater than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, or 8 μmol/m².

In one or more embodiments, the chromatographic stationary phase exhibits a retention change or drift of ≦5% over 30 days, ≦4% over 30 days, ≦3% over 30 days, ≦2% over 30 days, ≦1% over 30 days, ≦5% over 10 days, ≦4% over 10 days, ≦3% over 10 days, ≦2% over 10 days, ≦1% over 10 days, ≦5% over 3 days, ≦4% over 3 days, ≦3% over 3 days, ≦2% over 3 days, ≦1% over 3 days, ≦5% over 30 runs, ≦4% over 30 runs, ≦3% over 30 runs, ≦2% over 30 runs, ≦1% over 30 runs, ≦5% over 10 runs, ≦4% over 10 runs, ≦3% over 10 runs, ≦2% over 10 runs, ≦1% over 10 runs, ≦5% over 3 runs, ≦4% over 3 runs, ≦3% over 3 runs, ≦2% over 3 runs, or ≦1% over 3 runs.

In some embodiments, the core material consists essentially of a silica material. Optionally, the core material consists essentially of an organic-inorganic hybrid material or a superficially porous material. In one or more embodiments, the core material consists essentially of an inorganic material with a hybrid surface layer, a hybrid material with an inorganic surface layer, a surrounded hybrid layer, or a hybrid material with a different hybrid surface layer. The stationary phase material can optionally be in the form of a plurality of particles, a monolith, or a superficially porous material. In some embodiments the stationary phase material does not have chromatographically enhancing pore geometry whereas in other embodiments the stationary phase material has chromatographically enhancing pore geometry. The stationary phase material can be in the form of a spherical material, non-spherical material (e.g., including toroids, polyhedron). In certain embodiments, the stationary phase material has a highly spherical core morphology, a rod shaped core morphology, a bent-rod shaped core morphology, a toroid shaped core morphology; or a dumbbell shaped core morphology. In certain embodiments, the stationary phase material has a mixture of highly spherical, rod shaped, bent rod shaped, toroid shaped, or dumbbell shaped morphologies.

In some embodiments, the stationary phase material has a surface area of about 25 to 1100 m²/g, a surface area of about 150 to 750 m²/g, or a surface area of about 300 to 500 m²/g. In some embodiments, the stationary phase material has a pore volume of about 0.2 to 2.0 cm³/g or about 0.7 to 1.5 cm³/g. In one or more embodiments, the stationary phase material has a micropore surface area of less than about 105 m²/g, less than about 80 m²/g, or less than about 50 m²/g. In some embodiments, the stationary phase material has an average pore diameter of about 20 to 1500 Å, about 50 to 1000 Å, about 60 to 750 Å, or about 65 to 200 Å. In some embodiments, the plurality of particles has sizes between about 0.2 and 100 microns, between about 0.5 and 10 microns, or between about 1.5 and 5 microns.

In one or more embodiments of any of the above aspects, X is a silica core or a silica-organic hybrid core. T can be polar and Q and T can have a combined surface coverage of ≧1.5 μmol/m². T can be non-polar and Q and T can have a combined surface coverage of ≧2.0 μmol/m²; T can be non-polar and Q and T can have a combined surface coverage of ≧2.0 μmol/m²; T can be polar and Q and T can have a combined surface coverage of ≧1.5 μmol/m². In some embodiments, the chromatographic stationary phase includes radially adjusted pores, non-radially adjusted pores, ordered pores, non-ordered pores, monodispersed pores, non-monodispersed pores, smooth surfaces, rough surfaces or combinations thereof.

The chromatographic stationary phase can be adapted for supercritical fluid chromatography, carbon dioxide based chromatography, normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, sub-critical fluid chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof.

In another aspect, the present invention provides a chromatographic stationary phase material represented by Formula 1: [X](W)_(a)(Q)_(b)(T)_(c) (Formula 1). X can be a chromatographic core material that is subject to retention drift or change under normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography conditions. W can be absent and/or can include hydrogen and/or can include hydroxyl on the surface of X. Q can be bound directly to X and can include a functional group that essentially prevents chromatographic interaction between an analyte, and X and W. T can be bound directly to X, and can include a functional group that essentially prevents chromatographic interactions between an analyte, and X and W. Q can chromatographically interact with the analyte, T can chromatographically interact with the analyte, or both Q and T can chromatographically interact with the analyte. Additionally, Q and T, together, can essentially eliminate chromatographic interaction between the analyte, and X and W.

In one or more embodiments of any of the above aspects, Q is hydrophobic, T is hydrophobic, or Q and T are hydrophobic.

In yet another aspect, the present invention provides a chromatographic stationary phase material for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography represented by Formula 1: [X](W)_(a)(Q)_(b)(T)_(c) (Formula 1). X can be a high purity chromatographic core composition having a surface including a silica core material, a metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof. W can be absent and/or can include hydrogen and/or can include hydroxyl on the surface of X. Q can be bound directly to X and can include a first hydrophilic, polar, ionizable, charged and/or hydrophilic functional group that chromatographically interacts with the analyte. T can be bound directly to X and can include a second hydrophilic, polar, ionizable, charged and/or hydrophilic functional group that can chromatographically interact with the analyte. Additionally, Q and T can essentially eliminate chromatographic interaction between the analyte, and X and W, thereby minimizing retention variation over time (drift) under chromatographic conditions utilizing low water concentrations.

In one or more embodiments of any of the above aspects, the chromatographic stationary phase material is adapted for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof.

In yet another aspect, the invention provides a column or apparatus for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The column or apparatus includes a housing having at least one wall defining a chamber having an entrance and an exit, as well as a stationary phase according to any embodiments of the present invention disposed therein. The devices can have preformed frits, frits generated by interconnected materials, or devices without frits. The housing and stationary phase can be adapted for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof.

In yet another aspect, the present invention provides a kit for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The kit includes a housing having at least one wall defining a chamber having an entrance and an exit, and a stationary phase according to any embodiments of the present invention disposed therein. The housing and stationary phase can be adapted for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. Additionally, instructions for performing normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof with the housing and stationary phase can be included.

In another aspect, the present invention provides a method for preparing a stationary phase material in accordance with the present invention. The method includes (1) reacting a first chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with a chromatographic core material; and reacting a second chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with the chromatographic core material; (2) reacting a first chemical agent comprising one or more hydrophobic groups with a chromatographic core material; and reacting a second chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with the chromatographic core material; or (3) reacting a first chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with a chromatographic core material; and reacting a second chemical agent comprising one or more hydrophobic functional groups with the chromatographic core material, thereby producing a stationary phase material in accordance with the present invention.

In one or more embodiments, Q is derived from a reagent having one of the following structures:

In one or more embodiments, T is derived from a reagent having one of the following structures:

In yet another aspect, the present invention provides a method for mitigating or preventing retention drift or change in normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The method includes chromatographically separating a sample using a chromatographic device including a chromatographic stationary phase according to any embodiment of the present invention, thereby mitigating or preventing retention drift or change.

In one or more embodiments, mitigating or preventing retention drift or change includes a retention drift or change of ≦5% over 30 days, ≦4% over 30 days, ≦3% over 30 days, ≦2% over 30 days, ≦1% over 30 days, ≦5% over 10 days, ≦4% over 10 days, ≦3% over 10 days, ≦2% over 10 days, ≦1% over 10 days, ≦5% over 3 days, ≦4% over 3 days, ≦3% over 3 days, ≦2% over 3 days, ≦1% over 3 days, ≦5% over 30 runs, ≦4% over 30 runs, ≦3% over 30 runs, ≦2% over 30 runs, ≦1% over 30 runs, ≦5% over 10 runs, ≦4% over 10 runs, ≦3% over 10 runs, ≦2% over 10 runs, ≦1% over 10 runs, ≦5% over 3 runs, ≦4% over 3 runs, ≦3% over 3 runs, ≦2% over 3 runs, or ≦1% over 3 runs. In some embodiments, mitigating or preventing retention drift or change includes substantially eliminating the effect of alkoxylation and/or dealkoxylation of the chromatographic material on retention.

Additionally, Q can include one of the following structures:

R⁵ and R⁶ can independently represent methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group. R⁸ and R⁹ can be independently selected from methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, a zwitterion group, or a carbonyl, cyano, hydroxyl, fluoro, trifluoro, substituted aryl, ester, ether, amide, carbamate, urea, sulfoxide, nitro, nitroso, boronic acid, boronic ester, urea, thioether, sulfinyl, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocyclic, or triazole functionality. Het can represent a monocyclic or bicyclic heterocyclic or heteroaryl ring system including at least one nitrogen atom. B can represent an acidic ionizable modifier.

Additionally, Y can include one of the following structures:

The present invention is described in further detail by the figures and examples below, which are used only for illustration purposes and are not limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention can be more readily understood in the context of the following drawings and Detailed Description. It will be understood by a practitioner of ordinary skill in the art that the following drawings are not necessarily to scale, emphasis instead being placed on illustrating the inventive concepts of the present invention.

FIG. 1 shows a series of four chromatographic separations of analytes using silica 2-EP as a stationary phase.

FIG. 2 shows a series of four chromatographic separations of analytes using silica 2-EP that had previously been endcapped with TMS by treatment with chorotrimethylsilane as a stationary phase.

FIG. 3 shows the retention drift or change of the six components of the QC mix over 25 consecutive injections for a supercritical fluid chromatography apparatus using the silica 2-EP stationary phase.

FIG. 4 shows a series of four chromatographic separations of analytes using a BEH Technology™ (Waters Technologies Corporation, Milford Mass.) stationary phase.

FIG. 5 shows a series of four chromatographic separations of analytes using a BEH Technology™ (Waters Technologies Corporation, Milford Mass.) stationary phase that had previously been methoxylated by refluxing in methanol as a stationary phase.

FIG. 6 shows a series of four chromatographic separations of analytes using a bridged ethylene hybrid stationary phase to demonstrate the stability of the stationary phase.

FIG. 7 shows a graph of the percent original retention of an analyte over time for a various chromatography stationary phases.

DETAILED DESCRIPTION OF THE INVENTION

In various aspects and embodiments, the invention provides chromatographic materials for normal phase chromatography, supercritical fluid chromatography, carbon dioxide based chromatography, hydrophobic interaction liquid chromatography, hydrophilic interaction liquid chromatography, subcritical fluid chromatography, high pressure liquid chromatography, and solvated gas chromatography that mitigate or avoid retention drift or change while exhibiting useful overall retention, as well as corresponding apparatuses, kits, methods of manufacture, and methods of use.

The invention advantageously mitigates or avoids retention drift or change while exhibiting useful overall retention. For example, in SFC, retention drift or change can be (among various other theories) attributed to alkoxylation of solvent accessible silanols on the particle under the standard CO₂/MeOH mobile phase (and/or by other alcohol co-solvents) utilized for SFC. This is a problem, as users observe a change in the chromatography (e.g., retention time) obtained on their SFC system as the column ages, and again when a new, non-alkoxylated column is put on the system.

In various aspects and embodiments, the invention provides various solutions to such retention drift or change and related problems (e.g., retention, peak shape, and the like) through selection and/or modification of chromatographic materials.

Examples of three different routes to mitigate this problem include: (1) Functionalizing a chromatographic media with a small polar silane or other group that can functionalize the surface to effectively remove residual surface silanols. In various cases, using non-polar endcapping groups can significantly reduce the retentivity of the column—hence the requirement for an endcapping group containing a polar functionality. This group can be introduced, for example, via standard bonding chemistry. (2) Use of a base particle material other than a silica based material, or utilizing a silica based material with reduced silanol acidity. Alternatively, use of significantly higher surface area materials, bonded with a ligand such as 2-ethylpyridine, which is subsequently endcapped. (3) Alkoxylation of the chromatographic media prior to use. This can involve refluxing the media in an alcohol of choice, with or without the presence of a catalyst. Care should be taken to avoid aqueous solutions following this alkoxylation. These and other embodiments of the present invention are described in further detail below.

DEFINITIONS

In various aspects and embodiments, the invention provides for mitigating or preventing retention drift or change. “Retention drift” or “retention change” can include an undesired difference in elution time between chromatographic runs or experiments (e.g., in run 1, peak x elutes at time y, but in run 1+n, peak x elutes at time z). Thus, retention drift or change can result in undesired effects including experimental noise, irreproducibility, or failure. Accordingly, in a broad sense, mitigating or preventing retention drift or change includes addressing or counteracting an undesired difference in elution times between chromatographic runs, to the extent that the chromatographic experiment provides and chromatographically acceptable result.

In some embodiments, mitigating or preventing retention drift or change is not constant an absolute or constant value. For example, the amount of retention drift or change the can occur while still achieving a chromatographically acceptable result can vary depending upon the acceptable error or variance in a given experiment, the complexity of a sample (e.g., number and/or separation of peaks). The amount of retention drift or change the can occur while still achieving a chromatographically acceptable result can vary depending upon the duration or required reproducibility a given experiment (e.g., if reproducibility is required over a greater number of runs, the allowable retention drift or change between runs can be smaller). Therefore, it should be clear that mitigating or preventing retention drift or change does not necessarily mean the absolute elimination of retention drift or change.

In some embodiments, mitigating or preventing retention drift or change can be quantified. For example, retention drift or change can be measured for a single peak, or averaged over a set of peaks. Retention drift or change can be measured over a given period of time or number of runs. Retention drift or change can be measured relative to a standard value, starting value, or between two or more given runs.

Furthermore, retention drift or change can be quantified by a standardized test. In the examples discussed below, the following standardized test was employed: The Average % Retention Change was calculated by taking the percent difference of the average absolute peak retentions measured from the day 3, 10 or 30 chromatographic tests from the average absolute peak retentions measured on the day one chromatographic test. For each day tested, the columns were equilibrated under Mix1 test conditions for 20 minutes followed by three injections of Mix1 and then equilibrated under Mix2 Test conditions for 10 minutes, followed by three injections of Mix2.

The parameters for this standardized test are: Co-Solvent Mix1=5% methanol; Sample Mix1=3-benzoylpyridine (0.1 mg/mL); Co-Solvent Mix2=10% methanol; Sample Mix2=caffeine, thymine, papaverine, prednisolone, sulfanilamide (0.2 mg/mL each); Column Dimension=2.1×150 mm; Flow Rate=1.0 mL/min; Column Temperature=50° C.; Back Pressure=1800 psi; Detector=Waters Technologies Corporation ACQUITY PDA™ with SFC Flow Cell; Detector Setting=254 nm 40 spec/sec; Weak Needle Wash=iso-propanol; and Injection=1.0 μL (2.0 μL loop with PLUNO injection mode).

In accordance with this standardized test, mitigating or preventing retention drift or change can comprises a retention drift or change of ≦5% over 30 days, ≦4% over 30 days, ≦3% over 30 days, ≦2% over 30 days, ≦1% over 30 days, ≦5% over 10 days, ≦4% over 10 days, ≦3% over 10 days, ≦2% over 10 days, ≦1% over 10 days, ≦5% over 3 days, ≦4% over 3 days, ≦3% over 3 days, ≦2% over 3 days, ≦1% over 3 days, ≦5% over 30 runs, ≦4% over 30 runs, ≦3% over 30 runs, ≦2% over 30 runs, ≦1% over 30 runs, ≦5% over 10 runs, ≦4% over 10 runs, ≦3% over 10 runs, ≦2% over 10 runs, ≦1% over 10 runs, ≦5% over 3 runs, ≦4% over 3 runs, ≦3% over 3 runs, ≦2% over 3 runs, or ≦1% over 3 runs.

In other embodiments, mitigating or preventing retention drift or change can comprises a retention drift or change of ≦5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1% over 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days (or runs).

“High Purity” or “high purity chromatographic material” includes a material which is prepared form high purity precursors. In certain aspects, high purity materials have reduced metal contamination and/or non-diminished chromatographic properties including, but not limited to, the acidity of surface silanols and the heterogeneity of the surface.

“Chromatographic surface” includes a surface which provides for chromatographic separation of a sample. In certain aspects, the chromatographic surface is porous. In some aspects, a chromatographic surface can be the surface of a particle, a superficially porous material or a monolith. In certain aspects, the chromatographic surface is composed of the surface of one or more particles, superficially porous materials or monoliths used in combination during a chromatographic separation. In certain other aspects, the chromatographic surface is non-porous.

“Ionizable modifier” includes a functional group which bears an electron donating or electron withdrawing group. In certain aspects, the ionizable modifier contains one or more carboxylic acid groups, amino groups, imido groups, amido groups, pyridyl groups, imidazolyl groups, ureido groups, thionyl-ureido groups or aminosilane groups, or a combination thereof. In other aspects, the ionizable modifier contains a group bearing a nitrogen or phosphorous atom having a free electron lone pair. In certain aspects, the ionizable modifier is covalently attached to the material surface and has an ionizable group. In some instances it is attached to the chromatographic material by chemical modification of a surface hybrid group.

“Hydrophobic surface group” includes a surface group on the chromatographic surface which exhibits hydrophobicity. In certain aspects, a hydrophobic group can be a carbon bonded phase such as a C₄ to C₁₈ bonded phase. In other aspects, a hydrophobic surface group can contain an embedded polar group such that the external portion of the hydrophobic surface maintains hydrophobicity. In some instances it is attached to the chromatographic material by chemical modification of a surface hybrid group. In other instances the hydrophobic group can be C₄-C₃₀, embedded polar, chiral, phenylalkyl, or pentafluorophenyl bonding and coatings.

“Chromatographic core” includes a chromatographic material, including but not limited to an organic material such as silica or a hybrid material, as defined herein, in the form of a particle, a monolith or another suitable structure which forms an internal portion of the materials of the invention. In certain aspects, the surface of the chromatographic core represents the chromatographic surface, as defined herein, or represents a material encased by a chromatographic surface, as defined herein. The chromatographic surface material can be disposed on or bonded to or annealed to the chromatographic core in such a way that a discrete or distinct transition is discernible or can be bound to the chromatographic core in such a way as to blend with the surface of the chromatographic core resulting in a gradation of materials and no discrete internal core surface. In certain embodiments, the chromatographic surface material can be the same or different from the material of the chromatographic core and can exhibit different physical or physiochemical properties from the chromatographic core, including, but not limited to, pore volume, surface area, average pore diameter, carbon content or hydrolytic pH stability.

“Hybrid,” including “hybrid inorganic/organic material,” includes inorganic-based structures wherein an organic functionality is integral to both the internal or “skeletal” inorganic structure as well as the hybrid material surface. The inorganic portion of the hybrid material can be, e.g., alumina, silica, titanium, cerium, or zirconium or oxides thereof, or ceramic material. “Hybrid” includes inorganic-based structures wherein an organic functionality is integral to both the internal or “skeletal” inorganic structure as well as the hybrid material surface. As noted above, exemplary hybrid materials are shown in U.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913, the contents of which are incorporated herein by reference in their entirety.

The term “alicyclic group” includes closed ring structures of three or more carbon atoms. Alicyclic groups include cycloparaffins or naphthenes which are saturated cyclic hydrocarbons, cycloolefins, which are unsaturated with two or more double bonds, and cycloacetylenes which have a triple bond. They do not include aromatic groups. Examples of cycloparaffins include cyclopropane, cyclohexane and cyclopentane. Examples of cycloolefins include cyclopentadiene and cyclooctatetraene. Alicyclic groups also include fused ring structures and substituted alicyclic groups such as alkyl substituted alicyclic groups. In the instance of the alicyclics such substituents can further comprise a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, or the like.

The term “aliphatic group” includes organic compounds characterized by straight or branched chains, typically having between 1 and 22 carbon atoms. Aliphatic groups include alkyl groups, alkenyl groups and alkynyl groups. In complex structures, the chains can be branched or cross-linked. Alkyl groups include saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups and branched-chain alkyl groups. Such hydrocarbon moieties can be substituted on one or more carbons with, for example, a halogen, a hydroxyl, a thiol, an amino, an alkoxy, an alkylcarboxy, an alkylthio, or a nitro group. Unless the number of carbons is otherwise specified, “lower aliphatic” as used herein means an aliphatic group, as defined above (e.g., lower alkyl, lower alkenyl, lower alkynyl), but having from one to six carbon atoms. Representative of such lower aliphatic groups, e.g., lower alkyl groups, are methyl, ethyl, n-propyl, isopropyl, 2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl, tert-butyl, 3-thiopentyl and the like. As used herein, the term “nitro” means —NO₂; the term “halogen” designates —F, —Cl, —Br or —I; the term “thiol” means SH; and the term “hydroxyl” means —OH. Thus, the term “alkylamino” as used herein means an alkyl group, as defined above, having an amino group attached thereto. Suitable alkylamino groups include groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms. The term “alkylthio” refers to an alkyl group, as defined above, having a sulfhydryl group attached thereto. Suitable alkylthio groups include groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms. The term “alkylcarboxyl” as used herein means an alkyl group, as defined above, having a carboxyl group attached thereto. The term “alkoxy” as used herein means an alkyl group, as defined above, having an oxygen atom attached thereto. Representative alkoxy groups include groups having 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms, e.g., methoxy, ethoxy, propoxy, tert-butoxy and the like. The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogous to alkyls, but which contain at least one double or triple bond respectively. Suitable alkenyl and alkynyl groups include groups having 2 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms.

The term “alkyl” includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone, e.g., C₁-C₃₀ for straight chain or C₃-C₃₀ for branched chain. In certain embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone, e.g., C₁-C₂₀ for straight chain or C₃-C₂0 for branched chain, and more preferably 18 or fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms in their ring structure and more preferably have 4-7 carbon atoms in the ring structure. The term “lower alkyl” refers to alkyl groups having from 1 to 6 carbons in the chain and to cycloalkyls having from 3 to 6 carbons in the ring structure.

Moreover, the term “alkyl” (including “lower alkyl”) as used throughout the specification and claims includes both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Cycloalkyls can be further substituted, e.g., with the substituents described above. An “aralkyl” moiety is an alkyl substituted with an aryl, e.g., having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, e.g., phenylmethyl(benzyl).

The term “amino,” as used herein, refers to an unsubstituted or substituted moiety of the formula —NR_(a)R_(b), in which R_(a) and R_(b) are each independently hydrogen, alkyl, aryl, or heterocyclyl, or R_(a) and R_(b), taken together with the nitrogen atom to which they are attached, form a cyclic moiety having from 3 to 8 atoms in the ring. Thus, the term “amino” includes cyclic amino moieties such as piperidinyl or pyrrolidinyl groups, unless otherwise stated. An “amino-substituted amino group” refers to an amino group in which at least one of R_(a) and R_(b), is further substituted with an amino group.

The term “aromatic group” includes unsaturated cyclic hydrocarbons containing one or more rings. Aromatic groups include 5- and 6-membered single-ring groups which can include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine and the like. The aromatic ring can be substituted at one or more ring positions with, for example, a halogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, or the like.

The term “aryl” includes 5- and 6-membered single-ring aromatic groups that can include from zero to four heteroatoms, for example, unsubstituted or substituted benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine and the like. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl and the like. The aromatic ring can be substituted at one or more ring positions with such substituents, e.g., as described above for alkyl groups. Suitable aryl groups include unsubstituted and substituted phenyl groups. The term “aryloxy” as used herein means an aryl group, as defined above, having an oxygen atom attached thereto. The term “aralkoxy” as used herein means an aralkyl group, as defined above, having an oxygen atom attached thereto. Suitable aralkoxy groups have 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, e.g., O-benzyl.

The term “ceramic precursor” is intended include any compound that results in the formation of a ceramic material.

The term “chiral moiety” is intended to include any functionality that allows for chiral or stereoselective syntheses. Chiral moieties include, but are not limited to, substituent groups having at least one chiral center, natural and unnatural amino-acids, peptides and proteins, derivatized cellulose, macrocyclic antibiotics, cyclodextrins, crown ethers, and metal complexes.

The term “embedded polar functionality” is a functionality that provides an integral polar moiety such that the interaction with basic samples due to shielding of the unreacted silanol groups on the silica surface is reduced. Embedded polar functionalities include, but are not limited to carbonate, amide, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocyclic, triazole functionalities or carbamate functionalities such as disclosed in U.S. Pat. No. 5,374,755, and chiral moieties.

The language “chromatographically-enhancing pore geometry” includes the geometry of the pore configuration of the presently-disclosed materials, which has been found to enhance the chromatographic separation ability of the material, e.g., as distinguished from other chromatographic media in the art. For example, a geometry can be formed, selected or constructed, and various properties and/or factors can be used to determine whether the chromatographic separations ability of the material has been “enhanced,” e.g., as compared to a geometry known or conventionally used in the art. Examples of these factors include high separation efficiency, longer column life and high mass transfer properties (as evidenced by, e.g., reduced band spreading and good peak shape.) These properties can be measured or observed using art-recognized techniques. For example, the chromatographically-enhancing pore geometry of the present porous inorganic/organic hybrid materials is distinguished from the prior art materials by the absence of “ink bottle” or “shell shaped” pore geometry or morphology, both of which are undesirable because they, e.g., reduce mass transfer rates, leading to lower efficiencies.

Chromatographically-enhancing pore geometry is found in hybrid materials containing only a small population of micropores. A small population of micropores is achieved in hybrid materials when all pores of a diameter of about <34 Å contribute less than about 110 m²/g to the specific surface area of the material. Hybrid materials with such a low micropore surface area (MSA) give chromatographic enhancements including high separation efficiency and good mass transfer properties (as evidenced by, e.g., reduced band spreading and good peak shape). Micropore surface area (MSA) is defined as the surface area in pores with diameters less than or equal to 34 Å, determined by multipoint nitrogen sorption analysis from the adsorption leg of the isotherm using the BJH method. As used herein, the acronyms “MSA” and “MPA” are used interchangeably to denote “micropore surface area.”

The term “functionalizing group” includes organic functional groups which impart a certain chromatographic functionality to a chromatographic stationary phase.

The term “heterocyclic group” includes closed ring structures in which one or more of the atoms in the ring is an element other than carbon, for example, nitrogen, sulfur, or oxygen. Heterocyclic groups can be saturated or unsaturated and heterocyclic groups such as pyrrole and furan can have aromatic character. They include fused ring structures such as quinoline and isoquinoline. Other examples of heterocyclic groups include pyridine and purine. Heterocyclic groups can also be substituted at one or more constituent atoms with, for example, a halogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, or the like. Suitable heteroaromatic and heteroalicyclic groups generally will have 1 to 3 separate or fused rings with 3 to about 8 members per ring and one or more N, O or S atoms, e.g., coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl.

The term “metal oxide precursor” is intended include any compound that contains a metal and results in the formation of a metal oxide, e.g., alumina, silica, titanium oxide, zirconium oxide.

The term “monolith” is intended to include a collection of individual particles packed into a bed formation, in which the shape and morphology of the individual particles are maintained. The particles are advantageously packed using a material that binds the particles together. Any number of binding materials that are well known in the art can be used such as, for example, linear or cross-linked polymers of divinylbenzene, methacrylate, urethanes, alkenes, alkynes, amines, amides, isocyanates, or epoxy groups, as well as condensation reactions of organoalkoxysilanes, tetraalkoxysilanes, polyorganoalkoxysiloxanes, polyethoxysiloxanes, and ceramic precursors. In certain embodiments, the term “monolith” also includes hybrid monoliths made by other methods, such as hybrid monoliths detailed in U.S. Pat. No. 7,250,214; hybrid monoliths prepared from the condensation of one or more monomers that contain 0-99 mole percent silica (e.g., SiO₂); hybrid monoliths prepared from coalesced porous inorganic/organic particles; hybrid monoliths that have a chromatographically-enhancing pore geometry; hybrid monoliths that do not have a chromatographically-enhancing pore geometry; hybrid monoliths that have ordered pore structure; hybrid monoliths that have non-periodic pore structure; hybrid monoliths that have non-crystalline or amorphous molecular ordering; hybrid monoliths that have crystalline domains or regions; hybrid monoliths with a variety of different macropore and mesopore properties; and hybrid monoliths in a variety of different aspect ratios. In certain embodiments, the term “monolith” also includes inorganic monoliths, such as those described in G. Guiochon/J. Chromatogr. A 1168 (2007) 101-168.

The term “nanoparticle” is a microscopic particle/grain or microscopic member of a powder/nanopowder with at least one dimension less than about 100 nm, e.g., a diameter or particle thickness of less than about 100 nm (0.1 mm), which can be crystalline or noncrystalline. Nanoparticles have properties different from, and often superior to, those of conventional bulk materials including, for example, greater strength, hardness, ductility, sinterability, and greater reactivity among others. Considerable scientific study continues to be devoted to determining the properties of nanomaterials, small amounts of which have been synthesized (mainly as nano-size powders) by a number of processes including colloidal precipitation, mechanical grinding, and gas-phase nucleation and growth. Extensive reviews have documented recent developments in nano-phase materials, and are incorporated herein by reference thereto: Gleiter, H. (1989) “Nano-crystalline materials,” Prog. Mater. Sci. 33:223-315 and Siegel, R. W. (1993) “Synthesis and properties of nano-phase materials,” Mater. Sci. Eng. A168:189-197. In certain embodiments, the nanoparticles comprise oxides or nitrides of the following: silicon carbide, aluminum, diamond, cerium, carbon black, carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium, gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc, boron, and mixtures thereof. In certain embodiments, the nanoparticles of the present invention are selected from diamonds, zirconium oxide (amorphous, monoclinic, tetragonal and cubic forms), titanium oxide (amorphous, anatase, brookite and rutile forms), aluminum (amorphous, alpha, and gamma forms), and boronitride (cubic form). In particular embodiments, the nanoparticles of the present invention are selected from nano-diamonds, silicon carbide, titanium dioxide (anatase form), cubic-boronitride, and any combination thereof. Moreover, in particular embodiments, the nanoparticles can be crystalline or amorphous. In particular embodiments, the nanoparticles are less than or equal to 100 mm in diameter, e.g., less than or equal to 50 mm in diameter, e.g., less than or equal to 20 mm in diameter.

Moreover, it should be understood that the nanoparticles that are characterized as dispersed within the composites of the invention are intended to describe exogenously added nanoparticles. This is in contrast to nanoparticles, or formations containing significant similarity with putative nanoparticles, that are capable of formation in situ, wherein, for example, macromolecular structures, such as particles, can comprise an aggregation of these endogenously created.

The term “substantially disordered” refers to a lack of pore ordering based on x-ray powder diffraction analysis. Specifically, “substantially disordered” is defined by the lack of a peak at a diffraction angle that corresponds to a d value (or d-spacing) of at least 1 nm in an x-ray diffraction pattern.

“Surface modifiers” include (typically) organic functional groups which impart a certain chromatographic functionality to a chromatographic stationary phase. The porous inorganic/organic hybrid materials possess both organic groups and silanol groups which can additionally be substituted or derivatized with a surface modifier.

The language “surface modified” is used herein to describe the composite material of the present invention that possess both organic groups and silanol groups which can additionally be substituted or derivatized with a surface modifier. “Surface modifiers” include (typically) organic functional groups which impart a certain chromatographic functionality to a chromatographic stationary phase. Surface modifiers such as disclosed herein are attached to the base material, e.g., via derivatization or coating and later crosslinking, imparting the chemical character of the surface modifier to the base material. In one embodiment, the organic groups of a hybrid material react to form an organic covalent bond with a surface modifier. The modifiers can form an organic covalent bond to the material's organic group via a number of mechanisms well known in organic and polymer chemistry including but not limited to nucleophilic, electrophilic, cycloaddition, free-radical, carbene, nitrene, and carbocation reactions. Organic covalent bonds are defined to involve the formation of a covalent bond between the common elements of organic chemistry including but not limited to hydrogen, boron, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur, and the halogens. In addition, carbon-silicon and carbon-oxygen-silicon bonds are defined as organic covalent bonds, whereas silicon-oxygen-silicon bonds that are not defined as organic covalent bonds. A variety of synthetic transformations are well known in the literature, see, e.g., March, J. Advanced Organic Chemistry, 3rd Edition, Wiley, New York, 1985.

Chromatographic Materials

Chromatographic materials of the present invention can include those comprising a silica core material, metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof core material. The core material can be a high purity chromatographic core composition as discussed herein. Similarly, the chromatographic core material can be a regular, e.g., not high purity, version/analog/homolog of the high purity materials discussed herein.

Examples of suitable core materials include, but are not limited to, conventional chromatographic silica materials, metal oxide materials, inorganic-organic hybrid materials or a group of block copolymers thereof, ceramic, silicon oxide, silicon imidonitride, silicon nitride, silicon aluminum nitride, silicon diimide, and silicon oxynitride. Additional examples of suitable core materials (for use with or without modification) are described in US Pub. Nos. 2009/0127177, 2007/0135304, 2009/0209722, 2007/0215547, 2007/0141325, 2011/0049056, 2012/0055860, and 2012/0273404 as well as International Pub. No. WO2008/103423, which are incorporated herein by reference in their entirety.

The chromatographic core material can be in the form of discreet particles or can be a monolith. The chromatographic core material can be any porous material and can be commercially available or can be produced by known methods, such as those methods described in, for example, in U.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913, which are incorporated herein by reference in their entirety. In some embodiments, the chromatographic core material can be a non-porous core.

The composition of the chromatographic surface material and the chromatographic core material can be varied by one of ordinary skill in the art to provide enhanced chromatographic selectivity, enhanced column chemical stability, enhanced column efficiency, and/or enhanced mechanical strength. Similarly, the composition of the surrounding material provides a change in hydrophilic/lipophilic balance (HLB), surface charge (e.g., isoelectric point or silanol pK_(a)), and/or surface functionality for enhanced chromatographic separation. Furthermore, in some embodiments, the composition of the chromatographic material can also provide a surface functionality for available for further surface modification.

The ionizable groups and the hydrophobic surface groups of the chromatographic materials of the invention can be prepared using known methods. Some of the ionizable modifier reagents are commercially available. For example silanes having amino alkyl trialkoxysilanes, methyl amino alkyl trialkoxysilanes, and pyridyl alkyl trialkoxysilanes are commercially available. Other silanes such as chloropropyl alkyl trichlorosilane and chloropropyl alkyl trialkoxysilane are also commercially available. These can be bonded and reacted with imidazole to create imidazolyl alkyl silyl surface species, or bonded and reacted with pyridine to create pyridyl alkyl silyl surface species. Other acidic modifiers are also commercially available, including, but not limited to, sulfopropyltrisilanol, carboxyethylsilanetriol, 2-(carbomethoxy)ethylmethyldichlorosilane, 2-(carbomethoxy)ethyltrichlorosilane, 2-(carbomethoxy)ethyltrimethoxysilane, n-(trimethoxysilylpropyl)ethylenediamine, triacetic acid, (2-diethylphosphatoethyl)triethoxysilane, 2-(chlorosulfonylphenyl)ethyltrichlorosilane, and 2-(chloro sulfonylphenyl)ethyltrimethoxysilane.

It is known to one skilled in the art to synthesize these types of silanes using common synthetic protocols, including Grinard reactions and hydrosilylations. Products can be purified by chromatography, recrystallization or distillation.

Other additives such as isocyanates are also commercially available or can be synthesized by one skilled in the art. A common isocyanate forming protocol is the reaction of a primary amine with phosgene or a reagent known as triphosgene.

In one aspect, the present invention provides a chromatographic stationary phase material for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography. The chromatographic stationary phase is represented by Formula 1 [X](W)_(a)(Q)_(b)(T)_(c) (Formula 1). X can be a high purity chromatographic core composition having a surface including a silica core material, metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof. W can be absent and/or can include hydrogen and/or can include hydroxyl on the surface of X. Q can be bound directly to X and can include a first hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte. T can be bound directly to X and can include a second hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte. Additionally, Q and T can essentially eliminate chromatographic interaction between the analyte, and X and W, thereby minimizing retention variation over time (drift) under chromatographic conditions utilizing low water concentrations.

In another aspect, the present invention provides a chromatographic stationary phase represented by Formula 1:[X](W)_(a)(Q)_(b)(T)_(c) (Formula 1). X can be a chromatographic core material including a silica based, metal oxide based, or inorganic-organic hybrid based core surface. W can be absent and/or can include hydrogen and/or can include hydroxyl on the surface of X. Q can be bound directly to X and can include a first hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with an analyte. T can be bound directly to X and can include a second hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte. Additionally, Q and T can essentially eliminate chromatographic interactions between the analyte, and X and W.

In yet another aspect, the present invention provides a chromatographic stationary phase material represented by Formula 1:[X](W)_(a)(Q)_(b)(T)_(c) (Formula 1). X can be a chromatographic core material including a silica based, metal oxide based, or inorganic-organic hybrid based core surface. W can be absent and/or can include hydrogen and/or can includes hydroxyl on the surface of X. Q and T can each be independently bound directly to X. Q can include a hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with an analyte. X can include a non-polar group, or X can include a hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with an analyte. Q can include a non-polar group. Additionally, Q and T can essentially eliminate chromatographic interaction between the analyte, and X and W.

In one or more embodiments of any of the above aspects, Q can be represented by

In some embodiments, n¹ is an integer from 0-30; n² is an integer from 0-30; and n³=0 or 1, provided that when n³=0, n¹ is not 0. Each occurrence of R¹, R², R³ and R⁴ can independently represent hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, a protected or deprotected alcohol, a zwitterion, or a group Z. In some embodiments, Z represents a surface attachment group having the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein X is an integer from 1-3, y is an integer from 0-2, Z is an integer from 0-2, and x+y+z=3. Each occurrence of R⁵ and R⁶ can independently represent methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group. B¹ can represent a siloxane bond. In some embodiments, Z represents an attachment to a surface organofunctional hybrid group through a direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane linkage. In some embodiments, Z represents an adsorbed, surface group that is not covalently attached to the surface of the material. In some embodiments, Y is an embedded polar functionality. In some embodiments, A represents a hydrophilic terminal group. In some embodiments, A represents hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, or group Z. In some embodiments, A represents a functionalizable group.

In one or more embodiments of any of the above aspects, T is represented by:

In some embodiments, n¹ is an integer from 0-5; n² is an integer from 0-5; and n³=0 or 1, provided that when n³=0, n¹ is not 0. Each occurrence of R¹, R², R³ and R⁴ can independently represent hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, a protected or deprotected alcohol, a zwitterion, or a group Z. In some embodiments, Z represents a surface attachment group having the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein X is an integer from 1-3, y is an integer from 0-2, Z is an integer from 0-2, and x+y+z=3. Each occurrence of R⁵ and R⁶ can independently represent methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group and B¹ can represent a siloxane bond. In some embodiments, Z represents an attachment to a surface organofunctional hybrid group through a direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane linkage. In some embodiments, Z represents an adsorbed, surface group that is not covalently attached to the surface of the material. In some embodiments, Z represents a silyl ether bond. In some embodiments, Y is an embedded polar functionality. Additionally, in some embodiments, A represents a hydrophilic or ionizable terminal group; or in other embodiments A represents hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, or group Z.

In one or more embodiments of any of the above aspects, the chromatographic stationary T is one of the following structures:

In one or more embodiments, R⁵ and R⁶ independently represent methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group. Additionally, in some embodiments, A, A₁, and A₂ are independently selected hydrophilic/ionizable groups—including cyano, hydroxyl, fluoro, trifluoro, substituted aryl, ester, ether, amide, carbamate, urea, sulfoxide, nitro, nitroso, boronic acid, boronic ester, urea, thioether, sulfinyl, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocyclic, methyl, ethyl n-butyl, iso-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group, or triazole functionalities. In other embodiments, A, A₁ and A₂ are independently selected from non-polar groups—including methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, or lower alkyl. In yet other embodiments, A is independently selected from a hydrophilic/ionizable group, and A₁ is independently selected from a non-polar group and A₂ is independently selected from either a hydrophilic/ionizable group or from a non-polar group. In one or more embodiments, Q and T are different. In other embodiments, Q and T are the same. In some embodiments, the first functional group includes a diol, trimethoxysilyl ethyl pyridine, diethylaminotrimethoxysilane, a sulfur, nitrogen or oxygen based polar silanes carbonate, carbamate, amide, urea, ether, thioether, sulfinyl, sulfoxide, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocycle, or triazole. The functional group included by T can be an amine, trimethoxysilyl ethyl pyridine, diethylaminotrimethoxysilane, a sulfur, nitrogen or oxygen based polar silanes carbonate, carbamate, amide, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocycle, or triazole. Additionally, T, Q, or both Q and T can be achiral functional group adapted for a chiral separation.

In some embodiments, Q is one of the following structures:

In some embodiments, Q comprises a borate or nitro functional group.

In some embodiments, Z is a surface attachment group having the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—, wherein x is an integer from 1-3, y is an integer from 0-2, z is an integer from 0-2, and x+y+z=3. Each occurrence of R⁵ and R⁶ can independently represents methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group B¹ can represent a siloxane bond. In some embodiments, Z is an attachment to a surface organofunctional hybrid group through a direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane linkage. Additionally, in some embodiments, Z is an adsorbed, surface group that is not covalently attached to the surface of the material.

In some embodiments, the ratio b/c is about 0.05-75, 0.05-50, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90. Additionally, in one or more embodiments b=c, or b=0 and c>0 or b>0 and c=0. In some embodiments, a is ≧0. In some embodiments, the combined surface coverage is greater than about 1, 2, 3, 3.1, 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, or 8 μmol/m². The combined surface coverage can be greater than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, or 8 μmol/m².

In one or more embodiments, the chromatographic stationary phase exhibits a retention change or drift of ≦5% over 30 days, ≦4% over 30 days, ≦3% over 30 days, ≦2% over 30 days, ≦1% over 30 days, ≦5% over 10 days, ≦4% over 10 days, ≦3% over 10 days, ≦2% over 10 days, ≦1% over 10 days, ≦5% over 3 days, ≦4% over 3 days, ≦3% over 3 days, ≦2% over 3 days, ≦1% over 3 days, ≦5% over 30 runs, ≦4% over 30 runs, ≦3% over 30 runs, ≦2% over 30 runs, ≦1% over 30 runs, ≦5% over 10 runs, ≦4% over 10 runs, ≦3% over 10 runs, ≦2% over 10 runs, ≦1% over 10 runs, ≦5% over 3 runs, ≦4% over 3 runs, ≦3% over 3 runs, ≦2% over 3 runs, or ≦1% over 3 runs.

In some embodiments, the core material consists essentially of a silica material. Optionally, the core material consists essentially of an organic-inorganic hybrid material or a superficially porous material. In one or more embodiments, the core material consists essentially of an inorganic material with a hybrid surface layer, a hybrid material with an inorganic surface layer, a surrounded hybrid layer, or a hybrid material with a different hybrid surface layer. The stationary phase material can be in the form of a plurality of particles, a monolith, or a superficially porous material. In some embodiments the stationary phase material does not have chromatographically enhancing pore geometry whereas in other embodiments the stationary phase material has chromatographically enhancing pore geometry. The stationary phase material can be in the form of a spherical material, non-spherical material (e.g., including toroids, polyhedron). In certain embodiments, the stationary phase material has a highly spherical core morphology, a rod shaped core morphology, a bent-rod shaped core morphology, a toroid shaped core morphology; or a dumbbell shaped core morphology. In certain embodiments, the stationary phase material has a mixture of highly spherical, rod shaped, bent rod shaped, toroid shaped, or dumbbell shaped morphologies.

In some embodiments, the stationary phase material has a surface area of about 25 to 1100 m²/g, a surface area of about 150 to 750 m²/g, a surface area of about 300 to 500 m²/g, about 80 to 500 m²/g, or about 120 to 330 m²/g. In some embodiments, the stationary phase material has a pore volume of about 0.2 to 2.0 cm³/g, about 0.7 to 1.5 cm³/g, about 0.15 to 1.7 cm³/g; or about 0.5 to 1.3 cm³/g. In one or more embodiments, the stationary phase material has a micropore surface area of less than about 110 m²/g, of less than about 105 m²/g, less than about 80 m²/g, or less than about 50 m²/g. In some embodiments, the stationary phase material has an average pore diameter of about 20 to 1500 Å, about 50 to 1000 Å, about 60 to 750 Å, about 65 to 200 Å, about 100 to 750 Å; or about 150 to 500 Å. In some embodiments, the plurality of particles has sizes between about 0.2 and 100 microns, between about 0.5 and 10 microns, or between about 1.5 and 5 microns.

In one or more embodiments of any of the above aspects, X is a silica core or a silica-organic hybrid core. T can be polar and Q and T can have a combined surface coverage of ≧1.5 μmol/m². T can be non-polar and Q and T can have a combined surface coverage of ≧2.0 μmol/m²; T can be non-polar and Q and T can have a combined surface coverage of ≧2.0 μmol/m²; T can be polar and Q and T can have a combined surface coverage of ≧1.5 μmol/m². In some embodiments, the chromatographic stationary phase includes radially adjusted pores, non-radially adjusted pores, ordered pores, non-ordered pores, monodispersed pores, non-monodispersed pores, smooth surfaces, rough surfaces or combinations thereof.

The chromatographic stationary phase can be adapted for supercritical fluid chromatography, carbon dioxide based chromatography, normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, sub-critical fluid chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof.

In another aspect, the present invention provides a chromatographic stationary phase material represented by Formula 1: [X](W)_(a)(Q)_(b)(T)_(c) (Formula 1). X can be a chromatographic core material that is subject to retention drift or change under normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography conditions. W can be absent and/or can include hydrogen and/or can include hydroxyl on the surface of X. Q can be bound directly to X and can include a functional group that essentially prevents chromatographic interaction between an analyte, and X and W. T can be bound directly to X, and can include a functional group that essentially prevents chromatographic interactions between an analyte, and X and W. Q can chromatographically interact with the analyte, T can chromatographically interact with the analyte, or both Q and T can chromatographically interact with the analyte. Additionally, Q and T, together, can essentially eliminate chromatographic interaction between the analyte, and X and W.

In one or more embodiments of any of the above aspects, Q is hydrophobic, T is hydrophobic, or Q and T are hydrophobic.

In yet another aspect, the present invention provides a chromatographic stationary phase material for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography represented by Formula 1: [X](W)_(a)(Q)_(b)(T)_(c) (Formula 1). X can be a high purity chromatographic core composition having a surface including a silica core material, a metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof. W can be absent and/or can include hydrogen and/or can include hydroxyl on the surface of X. Q can be bound directly to X and can include a first hydrophilic, polar, ionizable, charged and/or hydrophobic functional group that chromatographically interacts with the analyte. T can be bound directly to X and can include a second hydrophilic, polar, ionizable, charged and/or hydrophobic functional group that can chromatographically interact with the analyte. Additionally, Q and T can essentially eliminate chromatographic interaction between the analyte, and X and W, thereby minimizing retention variation over time (drift) under chromatographic conditions utilizing low water concentrations.

In one or more embodiments of any of the above aspects, the chromatographic stationary phase material is adapted for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof.

In yet another aspect, the invention provides a column or apparatus for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The column or apparatus includes a housing having at least one wall defining a chamber having an entrance and an exit, as well as a stationary phase according to any embodiments of the present invention disposed therein. The devices can have preformed frits, frits generated by interconnected materials, or devices without frits. The housing and stationary phase can be adapted for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof.

In yet another aspect, the present invention provides a kit for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The kit includes a housing having at least one wall defining a chamber having an entrance and an exit, and a stationary phase according to any embodiments of the present invention disposed therein. The housing and stationary phase can be adapted for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. Additionally, instructions for performing normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof with the housing and stationary phase can be included.

In another aspect, the present invention provides a method for preparing a stationary phase material in accordance with the present invention. The method includes (1) reacting a first chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with a chromatographic core material; and reacting a second chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with the chromatographic core material; (2) reacting a first chemical agent comprising one or more hydrophobic groups with a chromatographic core material; and reacting a second chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with the chromatographic core material; or (3) reacting a first chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with a chromatographic core material; and reacting a second chemical agent comprising one or more hydrophobic functional groups with the chromatographic core material, thereby producing a stationary phase material in accordance with the present invention.

In one or more embodiments, Q is derived from a reagent having one of the following structures:

In one or more embodiments, T is derived from a reagent having one of the following structures:

In yet another aspect, the present invention provides a method for mitigating or preventing retention drift or change in normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The method includes chromatographically separating a sample using a chromatographic device including a chromatographic stationary phase according to any embodiment of the present invention, thereby mitigating or preventing retention drift or change.

In one or more embodiments, mitigating or preventing retention drift or change includes a retention drift or change of ≦5% over 30 days, ≦4% over 30 days, ≦3% over 30 days, ≦2% over 30 days, ≦1% over 30 days, ≦5% over 10 days, ≦4% over 10 days, ≦3% over 10 days, ≦2% over 10 days, ≦1% over 10 days, ≦5% over 3 days, ≦4% over 3 days, ≦3% over 3 days, ≦2% over 3 days, ≦1% over 3 days, ≦5% over 30 runs, ≦4% over 30 runs, ≦3% over 30 runs, ≦2% over 30 runs, ≦1% over 30 runs, ≦5% over 10 runs, ≦4% over 10 runs, ≦3% over 10 runs, ≦2% over 10 runs, ≦1% over 10 runs, ≦5% over 3 runs, ≦4% over 3 runs, ≦3% over 3 runs, ≦2% over 3 runs, or ≦1% over 3 runs. In some embodiments, mitigating or preventing retention drift or change includes substantially eliminating the effect of alkoxylation and/or dealkoxylation of the chromatographic material on retention.

In one or more aspects, the present invention provides a method of making a chromatographic stationary phase material for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The method includes selecting a high purity chromatographic core material having a surface comprising a silica core material, metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof. The method further includes reacting said core material with a first reagent, said first reagent comprising a first hydrophilic, polar, ionizable charged and/or hydrophilic functional group that chromatographically interacts with the analyte. The method yet further includes reacting said core material with a second reagent, said second reagent comprising a second hydrophilic, polar, ionizable charged and/or hydrophilic functional group that chromatographically interacts with the analyte; wherein said first and said second reagents eliminate chromatographic interactions between the analyte and the core material, thereby minimizing retention variation over time (drift) under chromatographic conditions utilizing low water concentrations.

Additionally, Q can include one of the following structures:

R⁵ and R⁶ can independently represent methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group. R⁸ and R⁹ can be independently selected from methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, a zwitterion group, or a carbonyl, cyano, hydroxyl, fluoro, trifluoro, substituted aryl, ester, ether, amide, carbamate, urea, sulfoxide, nitro, nitroso, boronic acid, boronic ester, urea, thioether, sulfinyl, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocyclic, or triazole functionality. Het can represent a monocyclic or bicyclic heterocyclic or heteroaryl ring system including at least one nitrogen atom. B can represent an acidic ionizable modifier.

Additionally, Y can include one of the following structures:

In certain other embodiments, the chromatographic material of the invention is non-porous. In another embodiment, the chromatographic material of the invention is hydrolytically stable at a pH of about 1 to about 14; at a pH of about 10 to about 14; or at a pH of about 1 to about 5.

In another aspect, the invention provides materials as described herein wherein the chromatographic material further comprises a nanoparticle or a mixture of more than one nanoparticles dispersed within the chromatographic surface.

In certain embodiments, the nanoparticle is present in <20% by weight of the nanocomposite, <10% by weight of the nanocomposite, or <5% by weight of the nanocomposite.

In other embodiments, the nanoparticle is crystalline or amorphous and can be silicon carbide, aluminum, diamond, cerium, carbon black, carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium, gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc, boron, oxides thereof, or a nitride thereof. In particular embodiments, the nanoparticle is a substance which comprises one or more moieties selected from the group consisting of nano-diamonds, silicon carbide, titanium dioxide, and cubic-boronitride. In other embodiments, the nanoparticles can be less than or equal to 200 nm in diameter, less than or equal to 100 nm in diameter, less than or equal to 50 nm in diameter, or less than or equal to 20 nm in diameter.

Preparation of Chromatographic Materials

The invention includes methods of producing the materials (e.g., the chromatographic materials) of the present invention. For instance, the invention can include methods of reacting a chromatographic stationary phase (e.g., silica particles) with a chemical reagent (e.g., methanol or TMS-C1) to chemically modify the surface of the stationary phase to mitigate the effects of retention drift or change. The invention also includes methods of producing chromatographic media that mitigate the effects of retention drift or change by changing the base particles (e.g., using a base particle other than silica). Furthermore, the invention also includes techniques such as refluxing silica particles in methanol (or a corresponding alcohol) to methoxylated (or alkoxylate) the surface of the silica particles (e.g., by means of a dehydration synthesis reaction).

Surface Modification of Chromatographic Core Materials:

In various aspects and embodiments, the invention includes modifying (e.g., endcapping) chromatographic core materials (e.g., as described above), to prepare chromatographic materials that mitigate or avoid retention drift or change. The concept of endcapping, as used herein, is understood to mean functionalizing a chromatographic core, for example with a small polar silane, or other functional group, thereby mitigating or avoiding retention drift or change. For example, endcapping can essentially prevent chromatographic interaction between an analyte and the chromatographic core (e.g., effectively eliminating a chromatographic effect of core surface silanols and/or alkoxylated silanols). In some cases, endcapping (e.g., using non-polar groups) can reduce the retentivity of the column. Therefore, in various embodiments surface modification of chromatographic core materials can include the use of hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte, to preserve or achieve a chromatographically useful overall retention. Such endcapping groups can be introduced, for example, via standard bonding chemistry.

In some embodiments, endcapping provides a permanent attachment. Accordingly, it is important to select an appropriate endcap for the chromatographic phase. In preferred embodiments, the chromatographic material will have chromatographically desirable properties (e.g., overall retention). Therefore in some embodiments it is important to select an endcap that has properties that can mimic the desirable (e.g., overall retention) properties of a conventional chromatographic material.

In various embodiments, the chemical properties of an endcapping group can be selected to achieve a desired effect. For example, one or more hydrophilic, polar, ionizable, and/or charged functional group can be used to achieve desired interactions with an analyte (e.g., chromatographically acceptable retention) and/or the mobile phase (e.g., repelling alcohols that might alkoxylate a chromatographic core surface). Likewise, endcap size and/or sterics can be selected to mask a core surface and/or effect a chiral separation.

Similarly, the concentration of endcaps can be varied. In some embodiments, larger and/or more strongly interacting endcaps can mitigate or avoid retention drift or change at lower concentrations (e.g., as compared to smaller endcaps). In other embodiments, coverage can be tailored for a desired property. For example, nonpolar endcaps can be used at lower coverage than polar endcaps (e.g., to maintain a desired retention). In various embodiments, endcapping can use one or more polar or nonpolar endcaps, or a combination thereof. In some embodiments, surface area of the chromatographic media is increased or decreased to compensate for decreased or increased retention due to the altered polarity of the endcaps.

Some exemplary endcaps that can be used in some embodiments are shown below. The endcaps can be silanes (e.g., chlorosilanes) that are attached to the chromatographic media by means of a chemical reaction (e.g., an S_(N)2 displacement reaction in the presence of a base). For instance, exemplary endcaps can include:

The above exemplary endcaps include masked polar functionality (e.g., alcohols). For instance, the first endcapping group above can yield an alcohol after hydrolysis of the acetate protecting group. Alternatively, the second endcapping group above can undergo epoxide ring opening with, for instance, water or an amine to form a diol or hydroxylamine moiety. Finally, treatment of a chromatographic surface with the third endcapping group above can automatically unmask a hydroxyl group upon cleavage of the Si—O bond as a result of the initial bonding with the chromatographic surface.

In some embodiments, the endcapping groups can already be charged prior to reacting with the chromatographic surface. For instance, the endcapping groups can have the zwitterionic structure given below:

In some embodiments, the endcapping groups can be fluorinated. For instance, the endcapping groups can be:

In some embodiments, the endcaps can include acidic groups. For instance, the endcapping groups can be:

In some embodiments, the endcapping groups can include alkyls. For instance, the endcapping groups can be —C₄H₉, TMFCL, HMDS, and the like.

In one or more aspects, the present invention provides a method of making a chromatographic stationary phase material for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The method includes selecting a high purity chromatographic core material having a surface comprising a silica core material, metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof. The method further includes reacting said core material with a first reagent, said first reagent comprising a first hydrophilic, polar, ionizable charged and/or hydrophilic functional group that chromatographically interacts with the analyte. The method yet further includes reacting said core material with a second reagent, said second reagent comprising a second hydrophilic, polar, ionizable charged and/or hydrophilic functional group that chromatographically interacts with the analyte; wherein said first and said second reagents eliminate chromatographic interactions between the analyte and the core material, thereby minimizing retention variation over time (drift) under chromatographic conditions utilizing low water concentrations.

The above method can be used to make any of the materials (e.g., chromatography stationary phase materials) as described herein. For instance, the methods of the present invention can include methods of reacting a chromatographic stationary phase (e.g., silica particles) with a chemical reagent (e.g., methanol or TMS-Cl, or any of the above reagents as described herein) to chemically modify the surface of the stationary phase to mitigate the effects of retention drift or change.

Use of Base Material(s) that Mitigate Retention Drift or Change:

In some embodiments, the invention uses different base material(s) that mitigate retention drift or change (e.g., without further chemical modification or endcapping). Such mitigation can be due to the material, for example, having less acidic silanols, a reduced number of silanols, a non-silica based material, or a combination thereof. The invention includes the materials themselves, methods of producing the materials, methods of using the materials (e.g., conducting SFC while mitigating retention drift or change), SFC equipment/apparatuses including the materials, kits including the materials with instructions for use thereof, and the like.

In some embodiments, the different base material(s) can be zirconia or alumina. In some embodiments, the base material(s) can be organic or polymer-based systems. For instance, an organic or polymeric base material can be highly crosslinked to avoid detectable bleed. An organic or polymeric base material can also be selected to avoid drift from the beginning due to a lack of acidic groups that can lead to retention drift or change in the first place.

In some embodiments, the base material is a coated inorganic material. Such materials can have the potential for immobilization. For instance, the chromatographic material can be an inorganic-organic hybrid material or silica coated with a hybrid material. In some embodiments, silanols are still present, but the material can have a less acidic silanol that would not exhibit drift. For instance, XTerra® coated particles (available from Waters Technologies Corporation, Milford Mass.) can reduce acidity. In some embodiments, the chromatographic media (including coated and uncoated media) can be selected to have more acidic silanols, for example to facilitate pre-alkoxylation and/or endcapping.

Alkoxylation of Chromatographic Media Prior to Use:

In some embodiments, chromatographic media (e.g., a chromatographic media described above) can be alkoxylated prior to use in a chromatographic setting (e.g., prior to being packed in a column, or prior to running a sample on a column). In one or more embodiments, the alkoxylation reaction is facilitated by refluxing the chromatographic media in the alcohol of choice. In some embodiments, a catalyst is used to facilitate alkoxylation of the chromatographic media, whereas in other embodiments, no catalyst is used. In preferred embodiments, care is taken after alkoxylation to avoid contacting the alkoxylated chromatographic media with aqueous solvents.

In some preferred embodiments, the alcohol chosen for alkoxylating the chromatographic media is selected based on the alcohol's relative resistance to subsequent hydrolysis (e.g., regeneration of a silanol —Si—OH bond and the corresponding alcohol upon addition of a molar equivalent of water). Without wishing to be bound by any particular theory, alcohols with more steric hindrance about the C—O bond can be less susceptible to hydrolysis. For instance, in some embodiments, the relative silanol dealkoxylation rate for various alcohols follows the order methanol>ethanol>isopropanol.

In some embodiments, the alcohols used for alkoxylating the chromatographic media include multiple hydroxyl functionalities. For instance, pentaerythritol (2,2-Bis(hydroxymethyl)1,3-propanediol), 2,2-dimethyl propanediol (neopentyl glycol) or tripentaerythritol can be used to alkoxylate the chromatographic media. In some embodiments, compounds that are labile (e.g., certain compounds with multiple alcohols) are not used to alkoxylate the chromatographic media.

Apparatuses

The present invention include various apparatuses (e.g., chromatographic columns, capillary and microfluidic devices, and systems for use thereof) including the chromatographic materials described herein. While several illustrative examples are discussed below, a practitioner of ordinary skill will understand that the present invention can contemplate a number of different embodiments, including but not limited to chromatographic columns, apparatuses, methods of use, or kits.

In some embodiments, the invention provides a column or apparatus for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The column or apparatus includes a housing having at least one wall defining a chamber having an entrance and an exit, as well as a stationary phase according to any embodiments of the present invention disposed therein. The devices can have preformed frits, frits generated by interconnected materials, or devices without frits. The housing and stationary phase can be adapted for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof.

Accordingly, the apparatus of the present invention can contain (e.g., be packed with) materials of the present invention (e.g., a chromatographic stationary phase such as a chemically modified stationary phase adapted to reduce or mitigate retention drift or change). Moreover, the apparatus of the present invention can be used to carry out the methods of the present invention as described herein.

In one embodiment, the present invention is in the form a packed column. The column can be packed with a stationary phase (e.g., chromatographic material) described herein. Such a column can be used to perform different types of chromatography (e.g., normal phase chromatography, supercritical fluid chromatography, carbon dioxide based chromatography, hydrophobic interaction liquid chromatography, hydrophilic interaction liquid chromatography, subcritical fluid chromatography, high pressure liquid chromatography, and solvated gas chromatography) while mitigating or avoiding retention drift or change.

The columns can be used in combination with existing chromatography platforms such as commercially available chromatography systems, including the Waters Alliance® HPLC system, Waters Acquity® system, or Waters UPC²® system. A column of the present invention can be used for a number of different mass throughputs (e.g., analytical scale chromatography, preparative scale chromatography) while mitigating the effects of retention drift or change. Likewise, the present invention can be embodies in capillary and microfluidic devices, and systems (e.g., commercially available and know to persons of ordinary skill in the art) for use thereof. The selection of columns, capillary, and microfluidic devices, and related systems will be readily understandable to person of ordinary skill in the art.

Methods of Use

In one or more embodiments, the present invention offers methods of use. For instance, a skilled practitioner will understand that the present invention contemplates methods of use of the chromatographic stationary phases described in the present disclosure to mitigate the effects of retention drift or change when performing a chromatographic separation. The present invention can include methods of using the described stationary phase for different types of chromatographic separations, such as capillary scale separations, analytical separations, preparative scale separations, or industrial processing scale separations. The present invention can include methods of use of the present invention in various modes of chromatography, (e.g., HPLC, UHPLC, or SFC) to reduce retention drift or change.

In one or more embodiments, the present invention provides a method for mitigating or preventing retention drift or change in normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The method includes chromatographically separating a sample using a chromatographic device including a chromatographic stationary phase according to any embodiment of the present invention, thereby mitigating or preventing retention drift or change.

The methods can be performed using a kit of the present invention as described herein. Additionally, the method can be performed with an apparatus, column or materials (e.g., chromatographic stationary phase) of the present invention as describe herein. Moreover, the methods of the present invention can be used to analyze (e.g., detect the presence, quantity, concentration and the like) analytes of the presentment invention.

The present invention can be used to separate and/or analyze a plurality of samples, including but not limited to small organic molecules, proteins, nucleic acids, lipids, fatty acids, carbohydrates, polymers, and the like. Similarly, the present invention can be used for the separation of small molecules, polar small molecules, analytes used in pharmaceuticals, biomolecules, antibodies, polymers and oligomers, sugars, glycan analysis, petrochemical analysis, lipid analysis, peptides, phosphopeptides, oligonucleotides, DNA, RNA, polar acids, polyaromatic hydrocarbons, food analysis, chemical analysis, bioanalysis, drugs of abuse, forensics, pesticides, agrochemicals, biosimilars, formulations.

In various embodiments, material in accordance with the present invention can have application in microbore columns for use on a SFC, HPLC, and/or UHPLC system.

In various embodiments, material in accordance with the present invention can have application fast equilibration columns, long lifetime columns, and SFC with water stable columns

Kits

The present invention provides a kit for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. The kit can include a housing having at least one wall defining a chamber having an entrance and an exit, and a stationary phase according to any embodiments of the present invention disposed therein. The housing and stationary phase can be adapted for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof. Additionally, instructions for performing normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof with the housing and stationary phase can be included.

Accordingly, the kit of the present invention can be used to implement the methods of the invention described herein. Additionally, the kits of the present invention can be used to analyze a variety of different samples and sample types, including those described herein below.

In one or more embodiments, the present invention can contemplate kits containing aspects of the present disclosure to reduce or mitigate the effects of retention drift or change. For instance, a kit can contain a chromatography column packed with a stationary phase media of the present disclosure. In some embodiments the packed column can be used directly in a standard chromatography system (e.g., a commercially available chromatography system such as a Waters Acquity® chromatography system). A kit can further contain instruction for use. Additionally, a kit can further contain stock samples of pure analyte for calibration of the instrument and/or confirmation of a substantial lack of retention drift or change. A kit can include any or all of the components described above (e.g., a stationary phase, a packed column, or a chromatography apparatus) to mitigate the effects of retention drift or change.

Analytes

Analytes amenable to chromatographic separation with the present invention can include essentially any molecule of interest, including, for example, small organic molecules, lipids, peptides, nucleic acids, synthetic polymers. The target analyte can be of interest, for example, in one or more of clinical chemistry, medicine, veterinary medicine, forensic chemistry, pharmacology, food industry, safety at work, and environmental pollution.

Clinical chemistry target analytes can include any molecule present in an organism (e.g., human body, animal body, fungi, bacterium, virus, and the like). For example, clinical chemistry target analytes include, but are not limited to, proteins, metabolites, biomarkers, and drugs.

Human medicine and veterinary medicine target analytes can include any molecule that can be used for the diagnosis, prophylaxis or treatment of a disease or condition in a subject. For example, human medicine and veterinary medicine target analytes include, but are not limited to, disease markers, prophylactic agents, or therapeutic agents.

Forensic chemistry target analytes can include any molecule present in a sample taken from the site of crime, such as a sample from a victim's body (e.g., tissue or fluid sample, hair, blood, semen, urine, and the like). For example, clinical chemistry target analytes include, but are not limited to, toxic agents, drugs and their metabolites, biomarkers, and identifying compounds.

Pharmacology target analytes can include any molecule that is a pharmaceutical or metabolite thereof or which can be used for the design, synthesis, and monitoring of drugs. For example, pharmacology target analytes include, but are not limited to, prophylactic and/or therapeutic agents, their prodrugs, intermediates and metabolites. Pharmacological analysis can include bioequivalence testing, for example, in connection with the approval, manufacturing, and monitoring of a generic drug.

Food industry and agricultural target analytes can include any molecule that is relevant for monitoring of the safety of foods, beverages, and/or other food industry/agricultural products. Examples of target analytes from the field of food industry include, but are not limited to, pathogen markers, allergens (e.g., gluten and nut proteins), and mycotoxins.

Target analytes can include polypeptides (e.g., polymers of naturally and/or non-naturally occurring amino acids such as Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Cys, Met, Ser, Thr, Tyr, His, Lys, Arg, Asp, Glu, Asn, Gln, selenocysteine, ornithine, citrulline, hydroxyproline, methyllysine, carboxyglutamate), peptides, polypeptides, proteins, glycoproteins, lipoproteins; peptide-nucleic acids; hormones (such as peptide hormones (e.g., TRH and vasopressin), as well as synthetic and industrial polypeptides.

Samples

In general, a sample is a composition including at least one target analyte (e.g., an analyte of the class or kind disclosed above, together with a matrix). Samples can include a solid, liquid, gas, mixture, material (e.g., of intermediary consistency, such as an extract, cell, tissue, organisms) or a combination thereof. In various embodiments, the sample is a bodily sample, an environmental sample, a food sample, a synthetic sample, an extract (e.g., obtained by separation techniques), or a combination thereof.

Bodily samples can include any sample that is derived from the body of an individual. In this context, the individual can be an animal, for example a mammal, for example a human. Other example individuals include a mouse, rat, guinea-pig, rabbit, cat, dog, goat, sheep, pig, cow, or horse. The individual can be a patient, for example, an individual suffering from a disease or being suspected of suffering from a disease. A bodily sample can be a bodily fluid or tissue, for example taken for the purpose of a scientific or medical test, such as for studying or diagnosing a disease (e.g., by detecting and/or identifying a pathogen or the presence of a biomarker). Bodily samples can also include cells, for example, pathogens or cells of the individual bodily sample (e.g., tumour cells). Such bodily samples can be obtained by known methods including tissue biopsy (e.g., punch biopsy) and by taking blood, bronchial aspirate, sputum, urine, faeces, or other body fluids. Exemplary bodily samples include humour, whole blood, plasma, serum, umbilical cord blood (in particular, blood obtained by percutaneous umbilical cord blood sampling (PUBS), cerebrospinal fluid (CSF), saliva, amniotic fluid, breast milk, secretion, ichor, urine, faeces, meconium, skin, nail, hair, umbilicus, gastric contents, placenta, bone marrow, peripheral blood lymphocytes (PBL), and solid organ tissue extract.

Environmental samples can include any sample that is derived from the environment, such as the natural environment (e.g., seas, soils, air, and flora) or the manmade environment (e.g., canals, tunnels, buildings). Exemplary environmental samples include water (e.g., drinking water, river water, surface water, ground water, potable water, sewage, effluent, wastewater, or leachate), soil, air, sediment, biota (e.g., soil biota), flora, fauna (e.g., fish), and earth mass (e.g., excavated material).

Food samples can include any sample that is derived from food (including beverages). Such food samples can be used for various purposes including, for example, (1) to check whether a food is safe; (2) to check whether a food contained harmful contaminants at the time the food was eaten (retained samples) or whether a food does not contain harmful contaminants; (3) to check whether a food contains only permitted additives (e.g., regulatory compliance); (4) to check whether it contains the correct levels of mandatory ingredients (e.g., whether the declarations on the label of the food are correct); or (5) to analyze the amounts of nutrients contained in the food. Exemplary food samples include edible products of animal, vegetable or synthetic origin (e.g., milk, bread, eggs, or meat), meals, drinks, and parts thereof, such as retain samples. Food samples can also include fruits, vegetables, pulses, nuts, oil seeds, oil fruits, cereals, tea, coffee, herbal infusions, cocoa, hops, herbs, spices, sugar plants, meat, fat, kidney, liver, offal, milk, eggs, honey, fish, and beverages.

Synthetic samples can include any sample that is derived from an industrial process. The industrial process can be a biological industrial process (e.g., processes using biological material containing genetic information and capable of reproducing itself or being reproduced in a biological system, such as fermentation processes using transfected cells) or a non-biological industrial process (e.g., the chemical synthesis or degradation of a compound such as a pharmaceutical). Synthetic samples can be used to check and monitor the progress of the industrial process, to determine the yield of the desired product, and/or measure the amount of side products and/or starting materials.

EXAMPLES Materials

All reagents were used as received unless otherwise noted. Those skilled in the art will recognize that equivalents of the following supplies and suppliers exist and, as such, the suppliers listed below are not to be construed as limiting.

Characterization

Those skilled in the art will recognize that equivalents of the following instruments and suppliers exist and, as such, the instruments listed below are not to be construed as limiting.

The % C values were measured by combustion analysis (CE-440 Elemental Analyzer; Exeter Analytical Inc., North Chelmsford, Mass.) or by Coulometric Carbon Analyzer (modules CM5300, CM5014, UIC Inc., Joliet, Ill.). Bromine and Chlorine content were determined by flask combustion followed by ion chromatography (Atlantic Microlab, Norcross, Ga.). The specific surface areas (SSA), specific pore volumes (SPV) and the average pore diameters (APD) of these materials were measured using the multi-point N₂ sorption method (Micromeritics ASAP 2400; Micromeritics Instruments Inc., Norcross, Ga.). The SSA was calculated using the BET method, the SPV was the single point value determined for P/P₀>0.98 and the APD was calculated from the desorption leg of the isotherm using the BJH method. The micropore surface area (MSA) was determined as the cumulative adsorption pore diameter data for pores <34 Å subtracted from the specific surface area (SSA). The median mesopore diameter (MMPD) and mesopore pore volume (MPV) were measured by Mercury Porosimetry (Micromeritics AutoPore II 9220 or AutoPore IV, Micromeritics, Norcross, Ga.). Skeletal densities were measured using a Micromeritics AccuPyc 1330 Helium Pycnometer (V2.04N, Norcross, Ga.). Particle sizes were measured using a Beckman Coulter Multisizer 3 analyzer (30 μm aperture, 70,000 counts; Miami, Fla.). The particle diameter (dp₅₀) was measured as the 50% cumulative diameter of the volume based particle size distribution. The width of the distribution was measured as the 90% cumulative volume diameter divided by the 10% cumulative volume diameter (denoted 90/10 ratio). Viscosity was determined for these materials using a Brookfield digital viscometer Model DV-II (Middleboro, Mass.). Measurements of pH were made with a Oakton pH100 Series meter (Cole-Palmer, Vernon Hills, Ill.) and were calibrated using Orion (Thermo Electron, Beverly, Mass.) pH buffered standards at ambient temperature immediately before use. Titrations were performed using a Metrohm 716 DMS Titrino autotitrator (Metrohm, Hersau, Switzerland), and are reported as milliequivalents per gram (mequiv/g). Multinuclear (¹³C, ²⁹Si) CP-MAS NMR spectra were obtained using a Bruker Instruments Avance-300 spectrometer (7 mm double broadband probe). The spinning speed was typically 5.0-6.5 kHz, recycle delay was 5 sec. and the cross-polarization contact time was 6 msec. Reported ¹³C and ²⁹Si CP-MAS NMR spectral shifts were recorded relative to tetramethylsilane using the external standards adamantane (¹³C CP-MAS NMR, δ 38.55) and hexamethylcyclotrisiloxane (²⁹Si CP-MAS NMR, δ −9.62). Populations of different silicon environments were evaluated by spectral deconvolution using DMFit software. [Massiot, D.; Fayon, F.; Capron, M.; King, I.; Le Calve, S.; Alonso, B.; Durand, J.-O.; Bujoli, B.; Gan, Z.; Hoatson, G. Magn. Reson. Chem. 2002, 40, 70-76]

TABLE 1 Base Particles Utilized Entry Material B1 Hybrid material (1.7 μm, 75 Å APD, 1.3 cm³/g TPV)¹ B2 Hybrid material (3.8 μm, 90 Å APD, 1.3 cm³/g TPV)¹ B3 Hybrid material (3.8 μm, 125 Å APD, 1.3 cm³/g TPV)¹ B4 Hybrid material with surrounding hybrid² B5 Hybrid material (1.7 μm, 125 Å APD, 0.7 cm³/g TPV)¹ ¹As described in U.S. Pat. No. 7,919,177, U.S. Pat. No. 7,223,473, U.S. Pat. No. 6,686,035 and WO2011084506 ²As described in US2012055860

TABLE 2 Silanes Utilized Entry Silane¹ S1 2-(2-pyridylethyl)trimethoxylsilane S2 2-(4-pyridylethyl)trimethoxylsilane S3 (N,N-diethyl-3-aminopropyl)trimethoxylsilane S4 phenethyltrimethoxysilane S5 3-methoxypropyltrimethoxysilane S6 3-cyanopropyldiisopropylchlorosilane S7 2-[2-[[3-(trimethoxysilyl)propyl]thio]ethyl]-pyridine S8 trimethylchlorosilane S9 triethylchlorosilane S10 tert-butyldimethylchlorosilane S11 triisopropylchlorosilane S12 (3,3,3-trifluoropropyl)-dimethylchlorosilane S13 3-cyanopropyldimethylchlorosilane S14 3-acetoxypropyldimethylchlorosilane S15 bis(3-cyanopropyl)dichlorosilane ¹From Gelest Inc., Morrisville, PA, Silar, Scotia, NY or Aldrich, Milwaukee, WI

Example I Representative Procedure for Primary Bonding

In a typical reaction, hybrid material (10 g) was refluxed in toluene (190 mL) using a Dean-Stark trap for 1 hour. Upon cooling, the desired silane and toluene (90 mL) were added to the flask. The mixture was stirred at room temperature for 1 hour, and then heated to reflux for 16 hours. The reaction was then cooled and the product was filtered and washed successively with toluene, acetone, 1:1 v/v acetone/water, and acetone (all solvents from Fisher Scientific). The particles were then slurried in a solution of acetone/0.1 M NH₄HCO₃ (60/40, v/v, 200 mL) and stirred for 20 hrs at 50° C. After cooling to room temperature, the particles were collected by filtration, and washed successively with 1:1 v/v acetone/water and acetone. The particles were dried overnight under vacuum at 80° C. Specific examples are presented in Table 3.

TABLE 3 List of Examples for Primary Bonding Base Material Silane Bonding Amount Silane Amount Coverage Example Particle (g) Used (g) (μmol/m²) 1A B2 200 S1 109 2.32 1B B2 15 S1 3.2 1.54 1C B3 20 S1 7.1 2.90 1D B1 20 S1 6.2 2.94 1E B2 15 S1 8.3 3.03 1F B2 25 S1 3.6 1.06 1G B2 80 S2 43.8 2.06 1H B2 60 S3 43.9 2.32 1I B2 15 S3 2.6 1.08 1J B2 15 S3 2.2 0.93 1K B2 15 S4 2.1 0.65 1L B2 15 S4 3.2 0.84 1M B2 15 S4 8.5 1.25 1N B2 15 S5 7.3 1.27 1O B2 20 S6 26.5 1.86 1P B2 30 S7 19.3 1.33 1Q B4 20 S1 7.1 3.41 1R B5 50 S1 8.6 2.07

Example 2 Representative Procedure for Primary Bonding with More than One Silane

In a typical reaction, hybrid material (10 g) was refluxed in toluene (190 mL) using a Dean-Stark trap for 1 hour. The slurry was then cooled to room temperature and silane A was added and the mixture heated to reflux for 1 hour. Upon cooling, silane B and toluene (90 mL) were added to the flask. The mixture was stirred at room temperature for 1 hour, and then heated to reflux for 16 hours. The reaction was then cooled and the product was filtered and washed successively with toluene, acetone, 1:1 v/v acetone/water, and acetone (all solvents from Fisher Scientific). The particles were then slurried in a solution of acetone/0.1 M NH₄HCO₃ (60/40, v/v, 200 mL) and stirred for 20 hrs at 50° C. After cooling to room temperature, the particles were collected by filtration, and washed successively with 1:1 v/v acetone/water and acetone. The particles were dried overnight under vacuum at 80° C. Specific examples are presented in Table 4.

TABLE 4 List of Examples for Primary Bonding with Mixed Silanes. Silanes Base Material Silane Silane Bonding Amount A Amount B Amount Coverage Example Particle (g) Used (g) Used (g) (μmol/m²) 2A B2 15 S1 0.52 S3 8.6 2.33 2B B2 20 S3 0.3 S1 11.1 2.35 2C B3 20 S3 0.72 S1 11.1 2.27 2D B1 20 S3 1.43 S1 11.1 2.26

Example 3 Representative Procedure for Secondary Bonding

In a typical example, the materials from Examples 1 and 2 (10 g) were refluxed in toluene (190 mL) using a Dean-Stark trap for 1 hour. The mixture was then cooled to room temperature and the desired silane and imidazole (Aldrich—Milwaukee, Wis., 1.2× molar excess to silane) were added. The mixture was refluxed for 4 hours, and then cooled to room temperature. The particles were collected by filtration, and washed successively with toluene, acetone, 1:1 v/v acetone/water, and acetone and then dried overnight under vacuum. Specific examples are presented in Table 5.

Additional Steps to Produce Material 3L

Material 3K (5.0 g) was heated at 60° C. for 6 hours in 40 mL of 0.1 M hydrochloric acid. After cooling the material was collected by filtration and washed with successively with water, and acetone and then dried at 80° C. overnight under vacuum.

TABLE 5 List of Examples for Secondary Bonding/Functionalization Base material Silane Bonding Overall Amount Silane Amount Coverage coverage¹ Example Particle (g) Used (g) (μmol/m²) (μmol/m²) 3A 1A 15 S6  20.3 0.64 2.96 3B 1A 20 S13 18.0 1.32 3.64 3C 1A 15 S13 1.5 0.57 2.89 3D 1A 20 S8  13.1 1.42 3.74 3E 1A 10 S8  1.0 0.62 2.94 3F 1A 10 S8  0.51 0.37 2.69 3G 1A 20 S9  18.2 1.08 2.40 3H 1A 20 S10 18.1 0.80 3.12 3I 1A 20 S11 23.2 0.49 2.81 3J 1A 20 S12 22.9 0.96 3.28 3K 1A 30 S14 21.0 0.86 3.18 3L 3K 5 See Experimental 0.30 2.62 3M 1A 5 S15 3.7 1.12 2.34 3N 1B 10 S6  13.5 0.89 2.43 3O 1C 10 S6  8.9 0.67 2.57 3P 1D 10 S6  7.5 0.64 2.58 3Q 1E 10 S6  13.3 0.54 3.57 3R 1F 10 S6  13.5 1.08 2.14 3S 1G 20 S6  27.1 0.71 2.77 3T 1G 20 S13 20.1 1.36 3.42 3U 1H 20 S6  27.1 0.37 2.69 3V 1H 20 S13 20.1 0.88 3.20 3W 1I 10 S6  13.5 0.77 1.85 3X 1J 10 S6  13.5 0.97 1.90 3Y 1K 7 S6  9.5 1.17 1.82 3Z 1L 10 S6  13.5 1.09 1.93 3AA 1M 10 S6  13.5 0.89 2.14 3BB 2A 10 S6  13.3 0.44 2.77 3CC 2B 20 S6  26.5 0.67 3.02 3DD 2C 20 S6  26.5 0.69 2.96 3EE 2D 20 S6  26.5 0.66 2.92 ¹Combined coverage (i.e., coverage from Example 1/Example 2 material + coverage from Example 3 material)

Example 4

The general procedure for bondings/functionalization of particles that is detailed in Examples 1, 2 and 3 is applied to modify the surface silanol groups of different porous materials. Included in this are monolithic, spherical, granular, superficially porous and irregular materials that are silica, hybrid inorganic/organic materials, hybrid inorganic/organic surface layers on hybrid inorganic/organic, silica, titania, alumina, zirconia, polymeric or carbon materials, and silica surface layers on hybrid inorganic/organic, silica, titania, alumina, zirconia or polymeric or carbon materials. Also includes are stationary phase materials in the form of a spherical material, non-spherical material (e.g., including toroids, polyhedron); stationary phase materials having a highly spherical core morphology, a rod shaped core morphology, a bent-rod shaped core morphology, a toroid shaped core morphology; or a dumbbell shaped core morphology; and stationary phase materials having a mixture of highly spherical, rod shaped, bent rod shaped, toroid shaped, or dumbbell shaped morphologies. Example hybrid materials are shown in U.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035, and 7,175,913 as well as International Publication No. WO2008/103423, the contents of which are hereby incorporated by reference in their entireties. Superficially porous particle include those describe in U.S. Pub. Nos. 2013/0112605, 2007/0189944, and 2010/061367, the contents of which are hereby incorporated by reference in their entireties. The particles size for spherical, granular or irregular materials can vary from 5-500 μm; more preferably 15-100 μm; more preferably 20-80 μm; more preferably 40-60 μm. The APD for these materials can vary from 30 to 2,000 Å; more preferably 40 to 200 Å; more preferably 50 to 150 Å. The SSA for these materials can vary from 20 to 1000 m²/g; more preferably 90 to 800 m²/g; more preferably 150 to 600 m²/g; more preferably 300 to 550 m²/g. The TPV for these materials can vary from 0.3 to 1.5 cm³/g; more preferably 0.5 to 1.4 cm³/g; more preferably 0.7 to 1.3 cm³/g. The macropore diameter for monolithic materials can vary from 0.1 to 30 μm, more preferably 0.5 to 25 μm, more preferably 1 to 20 μm.

Example 5 Retention Change in Materials from Examples 1, 2 and 3

The Average % Retention Change was calculated by taking the percent difference of the average absolute peak retentions measured from the day 3, 10 or 30 chromatographic tests from the average absolute peak retentions measured on the day one chromatographic test. For each day tested, the columns were equilibrated under Mix1 test conditions for 20 minutes followed by three injections of Mix1 and then equilibrated under Mix2 Test conditions for 10 minutes, followed by three injections of Mix2. Conditions are shown in Table 6. Results are shown in Tables 7 and 8 and in FIG. 7.

The % Less Retention was calculated by taking the percent difference of the day one average absolute peak retentions measured for Mix 1 and Mix 2 from the day one average absolute peak retentions measured for Mix 1 and Mix 2 on example 1A.

TABLE 6 Chromatographic Test Conditions for Retention Change Measurements Co-Solvent Mix1 5% methanol Sample Mix1 3-benzoylpyridine (0.1 mg/mL) Co-Solvent Mix2 10% methanol Sample Mix2 caffeine, thymine, papaverine, prednisolone, sulfanilamide Column Dimension 2.1 × 150 mm Flow Rate 1.0 mL/min Column Temperature 50° C. Back Pressure 1800 psi Detector Acquity PDA with SFC Flow Cell Detector Setting 254 nm 40 spec/sec Weak Needle Wash iso-propanol Injection 1.0 μL (2.0 μL loop with PLUNO injection mode)

TABLE 7 Retention Change in Materials From Examples 1 and 2 Over Time Average % Retention Change 3 Day 10 Day 30 Day Example Test Test Test 1A 7.5 11.5  15.7 1B 4.9 / 17.8 1C 5.8 / / 1D 8.2 / / 1F 5.5 14.1  14.3 1G 2.8 4.0  9.7 1H 2.0 3.5  3.7 1I 8.8 / 16.2 1J 5.8 / 15.5 1K 3.4 / / 1L 0.2 / / 1M 4.3 2.3  6.1 1O 0.9 / / 1P 5.4 3.8  8.1 1R 7.1 / / 2B 6.7 9.6 14.3 2C 6.5 10.7  14.9 2D 6.0 8.8 13.8 / indicates that this test was not performed for this material.

TABLE 8 Retention Change From Example 3 Materials Over Time % Less Average % Retention Retention Change than 3 Day 10 Day 30 Day Example Example Test Test Test 1A 3A 0.5 0.5 1.5 28.3 3B 0.0 0.0 0.1 67.4 3C 2.3 3.7 2.5 3D 0.6 3.4 3.6 75.0 3E 2.0 2.2 6.2 / 3F 4.3 6.1 10.1 / 3G 0.0 0.5 0.7 72.4 3H 2.0 1.1 0.9 64.6 3I 3.5 4.5 7.6 54.1 3J 2.1 3.3 8.3 82.2 3K 0.1 0.8 1.9 71.3 3M 0.6 0.3 5.2 42.7 3N 1.5 1.4 0.3 / 3O 1.8 1.9 3.5 / 3P 1.7 2.2 3.0 / 3Q 1.4 1.9 2.0 / 3R 0.4 0.2 1.3 / 3S 1.1 1.9 1.6 / 3T 1.4 1.6 2.2 / 3U 0.3 0.2 0.9 / 3V 1.2 / 1.7 / 3W 0.7 1.0 0.9 / 3X 0.5 1.3 0.2 / 3Y 2.6 / / / 3Z 3.1 / / / 3AA 1.1 0.5 2.4 / 3BB 0.2 1.8 0.8 / 3CC 0.6 2.0 0.6 / 3DD 0.4 2.3 2.8 / 3EE 0.7 1.3 0.7 / / indicates that this test was not performed for this material.

It is clear to one skilled in the art that the chromatographic retention change over time for materials that do not undergo treatment as in Example 3 (i.e., materials from Examples 1 and 2) is significantly larger than for those materials that do undergo treatment as in Example 3. This is particularly true for cases where a polar bonding/functionalization is utilized in Example 3.

Example 6 Silica 2-EP End Capping

An extended isocratic test was carried out to determine the effect endcapping on the retention drift or change and retention time of silica 2-EP (2-ethyl-pyridine) stationary phase. A Thar Investigator® WRC1-07 supercritical fluid chromatography apparatus equipped with Empower 2 with Service Pack E software was further equipped with a chromatography column containing a silica 2-EP stationary phase (4.6×150 mm). The apparatus was injected with a sample mix (QC mix) comprising flavone (0.1 mg/mL), thymine (0.2 mg/mL), papaverine (0.1 mg/mL), ketoprofen (0.2 mg/mL), prednisolone (0.5 mg/mL) and sulfanilamide (0.9 mg/mL) in MeOH (injection volume 2 μL; 5 μL loop with partial pick up injection mode). The injection needle was washed with methanol. The column was maintained at a temperature of 40° C. with a back pressure of 150 bar. The sample was eluted isocratically with a mobile phase comprising 10% methanol in carbon dioxide at a flow rate of 2.0 mL/min. The sample mixture was detected using a Waters® 2998 PDA detector at 220 nm (20 spec/sec). After a run time of 30 minutes, the column was flushed with methanol. The above procedure was repeated three additional times for a total of four runs using the bridged ethylene hybrid stationary phase. The retention differences between the six components of the QC mix from the first to the last injection were compared.

Next, the silica 2-EP stationary phase was endcapped with a trimethylsilyl (TMS) group. The chromatographic conditions and system were identical to those above except that the chromatography column was packed with the 2-EP silica endcapped with TMS. As above, the procedure was carried out a total of four times using the QC mix and eluting against the TMS-endcapped 2-EP silica. The retention differences between the six components of the QC mix from the first to the last injection were compared.

The results are shown in FIGS. 1 and 2. As shown in FIG. 1, the silica 2-EP chromatographic media shows a 5.8% average loss in retention time. Alternatively, the TMS endcapped silica 2-EP chromatographic media shows a −0.6% average loss in retention time. The endcapped silica 2-EP resulted in 33.4% less retention and a selectivity difference of 26 compared with the non-endcapped 2-EP silica. Accordingly, in some embodiments, endcapping can prevent retention shift, at least to negligible levels (e.g., −0.6%). However, in some embodiments, the prevention of retention shift was accompanied by a significant loss in overall retention (33.4%) and a selectivity difference (S=26) (Uwe Neue et al. J. Chromatography A, 1127 (2006) pp. 116-174) compared to the 2-EP silica. This can be an undesired side effect of the endcapping process.

FIG. 3 shows the retention drift or change of the six components of the QC mix over 25 consecutive injections for a supercritical fluid chromatography apparatus using the silica 2-EP stationary phase. The runs were carried out between an equilibration period of 20 minutes. As is seen from the figure, a steady reduction in retention times was observed.

Example 7 Methoxylation of Bridged Ethylene Hybrid Particles

An extended isocratic test was carried out to determine the effect of methoxylation on bridged ethylene hybrid particles. A Thar Investigator® WRC1-07 supercritical fluid chromatography apparatus equipped with Empower 2 with Service Pack E software was further equipped with a chromatography column containing a BEH Technology™ (Waters Technologies Corporation, Milford Mass.) stationary phase (4.6×150 mm). The apparatus was injected with a sample mix (QC mix) comprising flavone (0.1 mg/mL), thymine (0.2 mg/mL), papaverine (0.1 mg/mL), ketoprofen (0.2 mg/mL), prednisolone (0.5 mg/mL) and sulfanilamide (0.9 mg/mL) in MeOH (injection volume 2 μL; 5 μL loop with partial pick up injection mode). The injection needle was washed with methanol. The column was maintained at a temperature of 40° C. with a back pressure of 150 bar. The sample was eluted isocratically with a mobile phase comprising 10% methanol in carbon dioxide at a flow rate of 2.0 mL/min. The sample mixture was detected using a Waters® 2998 PDA detector at 220 nm (20 spec/sec). After a run time of 30 minutes, the column was flushed with methanol. The above procedure was repeated three additional times for a total of four runs using the bridged ethylene hybrid stationary phase. The retention differences between the six components of the QC mix from the first to the last injection were compared.

Next, the bridged ethylene hybrid stationary phase was methoxylated by refluxing in methanol. The chromatographic conditions and system were identical to those above except that the chromatography column was packed with the methoxylated bridged ethylene hybrid stationary phase particles. As above, the procedure was carried out a total of four times using the QC mix and eluting against the methoxylated stationary phase particles. The retention differences between the six components of the QC mix from the first to the last injection were compared.

The results are shown in FIGS. 4-6. As shown in FIG. 4, the stationary phase particles show a 0.5% average loss in retention time. Alternatively, as shown in in FIG. 5, the methoxylated stationary phase particles show a −0.1% average loss in retention time. As shown in in the figures when the stationary phase is methoxylated, significant shifts in retention and selectivity occur. Negligible retention shifts were recorded on fully methoxylated stationary phase. The fully methoxylated stationary phase resulted in 8.7% less retention and a significant selectivity difference of S=12.

FIG. 6 shows the stability of fully methoxylated stationary phase particles. Notably, methoxylated silica/hybrid surfaces are unstable in the presence of water. Also, when methoxylated sites are hydroxylated, retention and selectivity also shift.

Unless indicated otherwise, all techniques, including the use of kits and reagents, can be carried out according to the manufacturers' information, methods known in the art.

Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated, each individual value is incorporated into the specification as if it were individually recited. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, and instructions), are hereby incorporated by reference in their entirety.

The specification should be understood as disclosing and encompassing all possible permutations and combinations of the described aspects, embodiments, and examples unless the context indicates otherwise. One of ordinary skill in the art will appreciate that the invention can be practiced by other than the summarized and described aspect, embodiments, and examples, which are presented for purposes of illustration and not limitation. 

1. A chromatographic stationary phase material for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography represented by Formula 1: [X](W)_(a)(Q)_(b)(T)_(c)  Formula 1 wherein: X is a high purity chromatographic core composition having a surface comprising a silica core material, metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof; W is absent and/or includes hydrogen and/or includes hydroxyl on the surface of X; Q is bound directly to X and comprises a first hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte; T is bound directly to X and comprises a second hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte; and Q and T essentially eliminate chromatographic interaction between the analyte, and X and W, thereby minimizing retention variation over time (drift) under chromatographic conditions utilizing low water concentrations.
 2. A chromatographic stationary phase represented by Formula 1: [X](W)_(a)(Q)_(b)(T)_(c)  Formula 1 wherein: X is a chromatographic core material comprising a silica based, metal oxide based, or inorganic-organic hybrid based core surface; W is absent and/or includes hydrogen and/or includes hydroxyl on the surface of X; Q is bound directly to X and comprises a first hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with an analyte; T is bound directly to X and comprises a second hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with the analyte; and Q and T essentially eliminate chromatographic interaction between the analyte, and X and W.
 3. A chromatographic stationary phase material represented by Formula 1: [X](W)_(a)(Q)_(b)(T)_(c)  Formula 1 wherein: X is a chromatographic core material comprising a silica based, metal oxide based, or inorganic-organic hybrid based core surface; W is absent and/or includes hydrogen and/or includes hydroxyl on the surface of X; Q and T are each independently bound directly to X; Q comprises a hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with an analyte and X comprises a non-polar group, or X comprises a hydrophilic, polar, ionizable, and/or charged functional group that chromatographically interacts with an analyte and Q comprises a non-polar group; and Q and T essentially eliminate chromatographic interaction between the analyte, and X and W.
 4. The chromatographic stationary phase material of any one of claims 1-3, wherein Q is represented by:

wherein: n¹ an integer from 0-30; n² an integer from 0-30; n³=0 or 1, provided that when n³=0, n¹ is not 0; each occurrence of R¹, R², R³ and R⁴ independently represents hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, a protected or deprotected alcohol, a zwitterion, or a group Z; Z represents: a) a surface attachment group having the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si— wherein x is an integer from 1-3, y is an integer from 0-2, z is an integer from 0-2, and x+y+z=3, each occurrence of R⁵ and R⁶ independently represents methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group, and B¹ represents a siloxane bond; b) an attachment to a surface organofunctional hybrid group through a direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane linkage; or c) an adsorbed, surface group that is not covalently attached to the surface of the material; Y is an embedded polar functionality; and A represents i.) a hydrophilic terminal group; ii.) hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, or group Z; or iii.) a functionalizable group.
 5. The chromatographic stationary phase material of any one of claims 1-3, wherein T is represented by:

wherein: n¹ an integer from 0-5; n² an integer from 0-5; n³=0 or 1, provided that when n³=0, n¹ is not 0; each occurrence of R¹, R², R³ and R⁴ independently represents hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, a protected or deprotected alcohol, a zwitterion, or a group Z; Z represents: a) a surface attachment group having the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si— wherein x is an integer from 1-3, y is an integer from 0-2, z is an integer from 0-2, and x+y+z=3 each occurrence of R⁵ and R⁶ independently represents methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group; B¹ represents a siloxane bond b) an attachment to a surface organofunctional hybrid group through a direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane linkage; c) an adsorbed, surface group that is not covalently attached to the surface of the material; or d) a silyl ether bond Y is an embedded polar functionality; and A represents i.) a hydrophilic or ionizable terminal group; or ii.) hydrogen, fluoro, methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, or group Z.
 6. The chromatographic stationary phase material of any one of claims 1-3, wherein T comprises one of the following structures:

wherein R⁵ and R⁶ independently represent methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group, and wherein A, A₁, and A₂ are (1) independently selected from one of the following groups hydrophilic/ionizable groups, including cyano, hydroxyl, fluoro, trifluoro, substituted aryl, ester, ether, amide, carbamate, urea, sulfoxide, nitro, nitroso, boronic acid, boronic ester, urea, thioether, sulfinyl, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocyclic, methyl, ethyl, n-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group, or triazole functionalities, (2) independently selected from non-polar groups—including methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, or lower alkyl, and/or (3) independently selected from a hydrophilic/ionizable group, and A₁ is independently selected from a non-polar group and A₂ is independently selected from either a hydrophilic/ionizable group or from a non-polar group.
 7. The chromatographic stationary phase material of any one of claims 1-3, wherein Q and T are different.
 8. The chromatographic stationary phase material of any one of claims 1-3, wherein Q and T are the same.
 9. The chromatographic stationary phase material of any one of claims 1-3, wherein the first functional group comprises a diol, trimethoxysilyl ethyl pyridine, diethylaminotrimethoxysilane, a sulfur, nitrogen or oxygen based polar silanes carbonate, carbamate, amide, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbarnate, ethylene glycol, heterocycle, or triazole.
 10. The chromatographic stationary phase material of any one of claims 1-3, wherein the functional group comprised by T is an amine, trimethoxysilyl ethyl pyridine, diethylaminotrimethoxysilane, a sulfur, nitrogen or oxygen based polar silanes carbonate, carbamate, amide, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocycle, or triazole.
 11. The chromatographic stationary phase material of any one of claims 1-3, wherein T, Q, or both Q and T comprise a chiral functional group adapted for a chiral separation.
 12. The chromatographic stationary phase material of any one of claims 1-3, wherein Q comprises one of the following structures:

wherein Z comprises: a) a surface attachment group having the formula (B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si— wherein x is an integer from 1-3, y is an integer from 0-2, z is an integer from 0-2, and x+y+z=3 each occurrence of R⁵ and R⁶ independently represents methyl, ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, a protected or deprotected alcohol, or a zwitterion group; and B¹ represents a siloxane bond; b) an attachment to a surface organofunctional hybrid group through a direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane linkage; or c) an adsorbed, surface group that is not covalently attached to the surface of the material.
 13. The chromatographic stationary phase material of any one of claims 1-3, wherein b/c is about 0.05-75, 0.05-50, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90; or b=c; or b=0 and c>0; or b>0 and c=0.
 14. The chromatographic stationary phase material of any one of claims 1-3, wherein a is ≧0.
 15. The chromatographic stationary phase material of any one of claims 1-3, wherein the combined surface coverage is greater than about 1, 2, 3, 3.1, 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, or 8 μmol/m².
 16. The chromatographic stationary phase material of any one of claims 1-3, wherein the combined surface coverage is greater than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, or 8 μmol/m².
 17. The chromatographic stationary phase material of any one of claims 1-3, wherein the chromatographic stationary phase exhibits a retention change or drift of ≦5% over 30 days, ≦4% over 30 days, ≦3% over 30 days, ≦2% over 30 days, ≦1% over 30 days, ≦5% over 10 days, ≦4% over 10 days, ≦3% over 10 days, ≦2% over 10 days, ≦1% over 10 days, ≦5% over 3 days, ≦4% over 3 days, ≦3% over 3 days, ≦2% over 3 days, ≦1% over 3 days, ≦5% over 30 runs, ≦4% over 30 runs, ≦3% over 30 runs, ≦2% over 30 runs, ≦1% over 30 runs, ≦5% over 10 runs, ≦4% over 10 runs, ≦3% over 10 runs, ≦2% over 10 runs, ≦1% over 10 runs, ≦5% over 3 runs, ≦4% over 3 runs, ≦3% over 3 runs, ≦2% over 3 runs, or ≦1% over 3 runs.
 18. The chromatographic stationary phase material of any one of claims 1-3, wherein the core material consists essentially of a silica material.
 19. The chromatographic stationary phase material of any one of claims 1-3, wherein the core material consists essentially of an organic-inorganic hybrid material.
 20. The chromatographic stationary phase material of any one of claims 1-3, wherein the core material comprises a superficially porous material.
 21. The chromatographic stationary phase material of any one of claims 1-3, wherein the core material consists essentially of an inorganic material with a hybrid surface layer, a hybrid material with an inorganic surface layer, a surrounded hybrid layer, or a hybrid material with a different hybrid surface layer.
 22. The chromatographic stationary phase material of any one of claims 1-3, wherein the stationary phase material is in the form of a plurality of particles.
 23. The chromatographic stationary phase material of any one of claims 1-3, wherein the stationary phase material is in the form of a monolith.
 24. The chromatographic stationary phase material of any one of claims 1-3, wherein the stationary phase material is in the form of a superficially porous material.
 25. The chromatographic stationary phase material of any one of claims 1-3, wherein the stationary phase material does not have chromatographically enhancing pore geometry.
 26. The chromatographic stationary phase material of any one of claims 1-3, wherein the stationary phase material has chromatographically enhancing pore geometry.
 27. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has a surface area of about 25 to 1100 m²/g.
 28. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has a surface area of about 150 to 750 m²/g.
 29. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has a surface area of about 300 to 500 m²/g.
 30. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has a pore volume of about 0.2 to 2.0 cm³/g.
 31. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has a pore volume of about 0.7 to 1.5 cm³/g.
 32. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has a micropore surface area of less than about 105 m²/g.
 33. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has a micropore surface area of less than about 80 m²/g.
 34. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has a micropore surface area of less than about 50 m²/g.
 35. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has an average pore diameter of about 20 to 1500 Å.
 36. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has an average pore diameter of about 50 to 1000 Å.
 37. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has an average pore diameter of about 60 to 750 Å.
 38. The chromatographic stationary phase of claim 25 or 26, wherein the stationary phase material has an average pore diameter of about 65 to 200 Å.
 39. The chromatographic stationary phase of claim 25, wherein the plurality of particles have sizes between about 0.2 and 100 microns.
 40. The chromatographic stationary phase of claim 25, wherein the plurality of particles have sizes between about 0.5 and 10 microns.
 41. The chromatographic stationary phase of claim 25, wherein the plurality of particles have sizes between about 1.5 and 5 microns.
 42. The chromatographic stationary phase material of any one of claims 1-3, wherein X comprises a silica core or a silica-organic hybrid core and wherein (a) T is polar and Q and T have a combined surface coverage of ≧1.5 μmol/m²; (b) T is non-polar and Q and T have a combined surface coverage of ≧2.0 μmol/m²; (c) T is non-polar and Q and T have a combined surface coverage of ≧2.0 μmol/m²; (d) T is polar and Q and T have a combined surface coverage of ≧1.5 μmol/m².
 43. The chromatographic stationary phase material of any one of claims 1-3, wherein the chromatographic stationary phase comprises radially adjusted pores, non-radially adjusted pores, ordered pores, non-ordered pores, monodispersed pores, non-monodispersed pores, smooth surfaces, rough surfaces or combinations thereof.
 44. The chromatographic stationary phase material of any one of claims 1-3, wherein the chromatographic stationary phase is adapted for supercritical fluid chromatography.
 45. The chromatographic stationary phase material of any one of claims 1-3, wherein the chromatographic stationary phase is adapted for carbon dioxide based chromatography.
 46. The chromatographic stationary phase material of any one of claims 1-3, wherein the chromatographic stationary phase is adapted for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, sub-critical fluid chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof.
 47. A chromatographic stationary phase material represented by Formula 1: [X](W)_(a)(Q)_(b)(T)_(c)  Formula 1 wherein: X is a chromatographic core material that is subject to retention drift or change under normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography conditions; W is absent and/or includes hydrogen and/or includes hydroxyl on the surface of X; Q is bound directly to X and comprises a functional group that essentially prevents chromatographic interaction between an analyte, and X and W; T is bound directly to X, and comprises a functional group that essentially prevents chromatographic interaction between an analyte, and X and W; Q chromatographically interact with the analyte, T chromatographically interact with the analyte, or both Q and T chromatographically interact with the analyte; and Q and T, together, essentially eliminate chromatographic interaction between the analyte, and X and W.
 48. The chromatographic stationary phase material of claim 47, wherein Q is hydrophobic, T is hydrophobic, or Q and T are hydrophobic.
 49. A chromatographic stationary phase material for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography represented by Formula 1: [X](W)_(a)(Q)_(b)(T)_(c)  Formula 1 wherein: X is a high purity chromatographic core composition having a surface comprising a silica core material, metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof; W is absent and/or includes hydrogen and/or includes hydroxyl on the surface of X; Q is bound directly to X and comprises a first hydrophilic, polar, ionizable, charged and/or hydrophobic functional group that chromatographically interacts with the analyte; T is bound directly to X and comprises a second hydrophilic, polar, ionizable, charged and/or hydrophobic functional group that chromatographically interacts with the analyte; and Q and T essentially eliminate chromatographic interaction between the analyte, and X and W, thereby minimizing retention variation over time (drift) under chromatographic conditions utilizing low water concentrations.
 50. The chromatographic stationary phase of claim 2, 3, or 47, wherein the chromatographic stationary phase material is adapted for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, or a combination thereof.
 51. A column or apparatus for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography, hydrophobic interaction liquid chromatography, or a combination thereof comprising: a housing having at least one wall defining a chamber having an entrance and an exit, and a stationary phase according to any of claims 1-48 disposed therein, wherein the housing and stationary phase are adapted for normal phase chromatography, supercritical fluid chromatography, carbon dioxide based chromatography, or hydrophobic interaction liquid chromatography.
 52. A kit for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography, hydrophobic interaction liquid chromatography, or a combination thereof comprising: a housing having at least one wall defining a chamber having an entrance and an exit, and a stationary phase according to any of claims 1-48 disposed therein, wherein the housing and stationary phase are adapted for normal phase chromatography, supercritical fluid chromatography, carbon dioxide based chromatography, or hydrophobic interaction liquid chromatography; and instructions for performing normal phase chromatography, supercritical fluid chromatography, carbon dioxide based chromatography, or hydrophobic interaction liquid chromatography with the housing and stationary phase.
 53. A method for preparing a stationary phase according to any of claims 1-48 comprising: (1) reacting a first chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with a chromatographic core material; and reacting a second chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with the chromatographic core material; (2) reacting a first chemical agent comprising one or more hydrophobic groups with a chromatographic core material; and reacting a second chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with the chromatographic core material; or (3) reacting a first chemical agent comprising one or more hydrophilic, polar, ionizable, and/or charged functional groups with a chromatographic core material; and reacting a second chemical agent comprising one or more hydrophobic functional groups with the chromatographic core material, thereby producing a stationary phase material in accordance with any one of claims 1-48.
 54. The method of claim 53, wherein Q is derived from a reagent having one of the following structures:


55. The method of claim 53, wherein T is derived from a reagent having one of the following structures:


56. A method for mitigating or preventing retention drift or change in normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography, hydrophobic interaction liquid chromatography, or a combination thereof comprising: chromatographically separating a sample using a chromatographic device comprising a chromatographic stationary phase according to any one of claims 1-48, thereby mitigating or preventing retention drift or change.
 57. The method of claim 56, wherein mitigating or preventing retention drift or change comprises a retention drift or change of ≦5% over 30 days, ≦4% over 30 days, ≦3% over 30 days, ≦2% over 30 days, ≦1% over 30 days, ≦5% over 10 days, ≦4% over 10 days, ≦3% over 10 days, ≦2% over 10 days, ≦1% over 10 days, ≦5% over 3 days, ≦4% over 3 days, ≦3% over 3 days, ≦2% over 3 days, ≦1% over 3 days, ≦5% over 30 runs, ≦4% over 30 runs, ≦3% over 30 runs, ≦2% over 30 runs, ≦1% over 30 runs, ≦5% over 10 runs, ≦4% over 10 runs, ≦3% over 10 runs, ≦2% over 10 runs, ≦1% over 10 runs, ≦5% over 3 runs, ≦4% over 3 runs, ≦3% over 3 runs, ≦2% over 3 runs, or ≦1% over 3 runs.
 58. The method of claim 56, wherein mitigating or preventing retention drift or change comprises substantially eliminating the effect of alkoxylation and/or dealkoxylation of the chromatographic material on retention.
 59. A method of making a chromatographic stationary phase material for normal phase chromatography, high-pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, sub-critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography, hydrophobic interaction liquid chromatography, or a combination thereof comprising: (a) selecting a high purity chromatographic core material having a surface comprising a silica core material, metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof; (b) reacting said core material with a first reagent, said first reagent comprising a first hydrophilic, polar, ionizable charged and/or hydrophilic functional group that chromatographically interacts with the analyte; (c) reacting said core material with a second reagent, said second reagent comprising a second hydrophilic, polar, ionizable charged and/or hydrophilic functional group that chromatographically interacts with the analyte; wherein said first and said second reagents eliminate chromatographic interactions between the analyte and the core material, thereby minimizing retention variation over time (drift) under chromatographic conditions utilizing low water concentrations.
 60. The method of claim 59, wherein the stationary phase material is in accordance with any one of claims 1-48. 